Eukaryotic Cells with Artificial Endosymbionts for Multimodal Detection

ABSTRACT

The present invention is directed generally to eukaryotic cells comprising single-celled organisms that are introduced into the eukaryotic cell through human intervention and which transfer to daughter cells of the eukaryotic cell, and methods of introducing such single-celled organisms into eukaryotic cells. The invention provides single-celled organisms that introduce a phenotype to eukaryotic cells that is maintained in daughter cells. The invention additionally provides eukaryotic cells containing magnetic bacteria. The invention further provides eukaryotic cells engineered with single-celled organisms to allow for multimodal observation of the eukaryotic cells. Each imaging method (or modality) allows the visualization of different aspects of anatomy and physiology, and combining these allows the imager to learn more about the subject being imaged.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/263,144, filed on Sep. 12, 2016, which is a continuation of U.S.application Ser. No. 15/079,453, filed on Mar. 24, 2016, now U.S. Pat.No. 9,446,154, which is a continuation of U.S. application Ser. No.14/689,987 filed Apr. 17, 2015, now U.S. Pat. No. 9,315,780, which is acontinuation of U.S. application Ser. No. 14/332,373 filed Jul. 15,2014, now U.S. Pat. No. 9,023,612, which is a continuation-in-part ofU.S. application Ser. No. 13/838,717, filed Mar. 15, 2013, now U.S. Pat.No. 8,828,681, which is a continuation-in-part of U.S. application Ser.No. 13/374,799, filed on Jan. 13, 2012, now U.S. Pat. No. 8,956,873, andwhich is a continuation-in-part of PCT/US2013/021414, filed on Jan. 14,2013, which is a continuation-in-part of U.S. application Ser. No.13/374,799, filed on Jan. 13, 2012, now U.S. Pat. No. 8,956,873, theentire contents of which applications are hereby incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to the field of endosymbiosis,eukaryotic cells engineered with artificial endosymbionts, andmagnetotactic bacteria. In particular, the invention providessingle-celled organisms such as artificial endosymbionts, includingmagnetotactic bacteria, eukaryotic cells to host those single-celledorganisms, methods of using eukaryotic cells containing single-celledorganisms, and methods of introducing the single-celled organisms intothe eukaryotic cells. The invention also provides eukaryotic cellsengineered with intracellular single-celled organism allowing multimodaldetection of the eukaryotic cells.

BACKGROUND OF THE INVENTION

Mitochondria, chloroplast and other membrane bound organelles addheritable functionalities, such as photosynthesis, to eukaryotic cells.Such organelles (identified by their vestigial circular DNA) arebelieved to be endosymbiotically derived.

Bacteria exist with a wide range of functionalities not present invarious eukaryotic cells. For example, in 1975 Blakemore identifiedmagnetotactic bacteria (MTB) that orient and swim along a geomagneticfield. (Blakemore, R.,“Magnetotactic bacteria,” Science 24:377-379(1975), which is incorporated by reference in its entirety for allpurposes). These magnetotactic bacteria produce magnetic structurescalled magnetosomes that are composed of magnetite (Fe₃O₄) or greigite(Fe₃S₄) enclosed by a lipid membrane. (Id.). A large number of MTBspecies have been identified since their initial discovery. (Id.).

Magnetotactic bacteria have been used to selectively bind to andseparate substances. (U.S. Pat. No. 4,677,067, incorporated by referenceherein in its entirety for all purposes). Additionally, attempts havebeen made to add magnetic functionality to cells through external tags.(Swiston, A. J. et al., “Surface Functionalization of Living Cells withMultilayer Patches,” Nano Lett. 8(12):4446-53 (2008); Vandsburger, M. H.et al., “MRI reporter genes: applications for imaging of cell survival,proliferation, migration and differentiation,” NMR Biomed. 26(7):872-84(2013); Ahrens, E. T. et al, “Tracking immune cells in vivo usingmagnetic resonance imaging,” Nature Rev Immunol. 13:755-763 (2013);which publications are incorporated by reference in its entirety for allpurposes). Bacterial magnetite has also been introduced into red bloodcells by cell fusion (Matsunaga, T. and Kamiya, S., (1988), In: Atsumi,K., Kotani, M., Ueno, S., Katila T., Williamsen, S. J. (Eds) 6thInternational Conference on Biomagnetisms, Tokyo Denki University Press,Tokyo, pp. 50-51 (1988), which is incorporated by reference in itsentirety for all purposes), and MTB have been introduced intogranulocytes and monocytes by phagocytosis. (Matsunaga, T. et al.,“Phagocytosis of bacterial magnetite by leucocytes,” AppliedMicrobiology and Biotechnology 31(4):401-405 (1989), which isincorporated by reference in its entirety for all purposes)). However,none of these alterations are heritable to daughter cells.

Currently there is a need in the imaging field for a multimodal probewhich can label and track eukaryotic cells with MRI or other types ofimaging techniques with minimal manipulation of the host cells. In someembodiments, it is an object of the present invention to provideeukaryotic cells containing a single-celled organism that is introducedinto the eukaryotic cell through human intervention which transfers todaughter cells of the eukaryotic cell, in particular through at leastfive cell divisions, and which maintains sufficient copy number in thedaughter cells so that a desired functionality introduced by thesingle-celled organism is maintained in the daughter cells. It isfurther an object of the present invention to provide eukaryotic hostcells containing artificial endosymbionts that are heritable to daughtercells and methods of uses of these eukaryotic cells. It is also anobject of the present invention to provide methods of introducingartificial endosymbionts into the cytosol of eukaryotic host cells. Itis another object of the present invention to provide eukaryotic cellswith a heritable magnetic phenotype. It is also an object of theinvention to provide methods of tracking, localizing, steering,controlling or damaging eukaryotic cells. It is another object of thepresent invention to provide eukaryotic cells containing a single-celledorganism that allows for multimodal detection of the eukaryotic cells.It is a further object of the present invention to provide eukaryotichost cells containing a single-celled organism such that multiplephenotypes are heritable to daughter cells.

SUMMARY OF THE INVENTION

The present invention relates to eukaryotic cells comprisingsingle-celled organisms, such as artificial endosymbionts, methods ofusing such eukaryotic cells, and methods of introducing suchsingle-celled organisms into eukaryotic cells. In one embodiment, thesingle-celled organism provides the eukaryotic cell with a desiredfunctionality. In one embodiment, the single-celled organisms areartificial endosymbionts heritable to daughter cells. In anotherembodiment, the artificial endosymbiont is a magnetotactic bacteriummaking magnetosomes and expressing another gene product that can bedetected. In one embodiment, the magnetotactic bacterium provides theeukaryotic cell with a magnetic functionality. In one embodiment, amethod of use is a method of detecting the eukaryotic cells. In anotherembodiment, a method of use is a method of magnetically manipulating ortargeting the eukaryotic cells. In another embodiment, a method of useis a method of damaging the eukaryotic cells. In one embodiment theeukaryotic cells have multiple phenotypes that allow for multimodaldetection, which may be introduced to the eukaryotic cell by thesingle-celled organism. In one embodiment, the multiple phenotypes areheritable to daughter cells of the eukaryotic cells. In one embodiment,the magnetotactic bacterium provides the eukaryotic cell with a magneticfunctionality and a light emissive or absorptive property and/or anacoustic property. In an embodiment, the single-celled organism producesa gene product that interacts with a gene product from the eukaryoticcell to produce a detectable signal. In one embodiment, the eukaryoticcell containing the single-celled organism is used in a method ofdetecting the eukaryotic cells.

In some embodiments, the artificial endosymbiont of the invention may bemodified by deleting, adding, and/or mutating at least one gene wherebythe artificial endosymbiont acquires a trait useful for endosymbiosis orbiotrophy. The genes to be mutated, added, and/or deleted in theartificial endosymbiont may be genes encoding components of theflagellar assembly and genes encoding enzymes for synthesizing essentialmacromolecules, such as amino acids, nucleotides, vitamins, andco-factors. In certain embodiments, the MTB may further be modified toexpress an antibiotic resistance gene or other selectable marker. Incertain embodiments, the genes localize artificial endosymbionts tospecific subcellular locations. In certain embodiments the genes provideenhanced or blocked entry of the artificial endosymbionts to specifichost cells. In other embodiments the gene suppresses or alters the hostimmune system response to the artificial endosymbiont or genes andproteins expressed from it.

In some embodiments the eukaryotic cells of the invention are mammalian,such as mouse, rat, rabbit, hamster, human, porcine, bovine, or canine.In another embodiment, the artificial endosymbiont is transmitted fromthe host cell to daughter progeny host cells. In another embodiment, themethod further comprises deleting, inserting, and/or mutating at leastone gene from the eukaryotic cell.

The single-celled organisms of the invention can be introduced intoeukaryotic cells by a number of methods known to those of skill in theart including, but not limited to, microinjection, natural phagocytosis,induced phagocytosis, macropinocytosis, other cellular internalizationprocesses, liposome fusion, erythrocyte ghost fusion, orelectroporation.

The invention also relates to methods of using multimodal detection tocharacterize the eukaryotic cell containing the single-celled organism.In the methods of the invention, eukaryotic cells with single-celledorganisms have multiple phenotypes that relate to the single-celledorganism. In an embodiment, the multiple phenotypes allow for detectionof the eukaryotic cells using multimodal observation methods. In anembodiment, multimodal detection is used for non-invasive in vivoimaging of the eukaryotic cells. Each method or modality of imaging canallow visualization of different aspects of anatomy and physiology, andcombining these provides greater information about the eukaryotic cellsin an in vivo environment.

In an embodiment, multimodal detection of the eukaryotic cell is usedfor simultaneously acquiring multiple forms of functional data about thecell, and/or enabling detection of the cell on a range of differentimaging devices.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows positive contrast generated with a T₁ pulse sequence over alog scale concentration up to ˜10⁸ MTB/mL for gfp⁺AMB-1 suspended inagar plugs using a 1.5T instrument to optimize and characterize theimaging properties.

FIG. 2A-B shows a blastula stage mouse embryo that has had one of itstwo cells at the 2-cell embryo stage microinjected with gfp⁻AMB-1. Theembryo is imaged with Leica SP2 AOBS spectral confocal invertedmicroscope surrounded by an environmental control chamber for live-cellimaging with 20×, 0.7 NA objective, and optical zoom of 3×. FIG. 2Ashows differential interference contrast (DIC) image and FIG. 2B shows agray scale fluorescence capture of the same image.

FIG. 3 shows the change of total embryo GFP fluorescence of four mouseembryos over time as measured by confocal microscopy. One of the twocells from the 2-cell stage of each embryo had been microinjected withgfp⁺AMB-1, and the total GFP fluorescence of each embryo was measuredbeginning at the 8-cell stage, 24 hours after microinjection.

FIGS. 4A-C show images taken of MDA-MB-231 human breast carcinoma cellscontaining gfp+ AMB-1. FIG. 4A shows a phase contrast image ofMDA-MB-231 human breast carcinoma cells containing gfp+ AMB-1. FIG. 4Bshows a fluorescence image of the same cells, demonstrating fluorescencesignal from the gfp+ AMB-1. FIG. 4C shows a T2w MRI image of a tubefilled with agarose (bottom half) and phosphate-buffered saline (tophalf), with a layer of MDA-MB-231 cells containing gfp+ AMB-1 in between(dark band). FIGS. 4D-F show phase contrast, fluorescence, and MRIimages of MDA-MB-231 cells without gfp+ AMB-1.

FIGS. 5A-C show MDA-MB-231 cells containing gfp+ AMB-1, suspended inagarose gel in a cylindrical 100 ml tube. FIG. 5A is an image of theMDA-MB-231 cells containing gfp+ AMB-1 as seen on visual inspection.FIG. 5B shows an MRI image of the MDA-MB-231 cells containing gfp+AMB-1. FIG. 5C shows a fluorescence image of the MDA-MB-231 cellscontaining gfp+ AMB-1.

FIGS. 6A-L show different cells without gfp+ AMB-1 compared to cellscontaining gfp+ AMB-1. Cells are imaged with an epifluorescencemicroscope and fluorescence imaging. iPS (induced pluripotent stemcells, FIGS. 6A and 6B), MDA-MB-231 (breast carcinoma cells, FIGS. 6Cand 6D), J774.2 cells (murine macrophage, FIGS. 6E and 6F), BJ (humanfibroblast, FIGS. 6G and 6H), HEP1 cells (human liver adenocarcinoma,FIGS. 6I and 6J), and MCF7 (human epithelial breast adenocarcinoma,FIGS. 6K and 6L) were used. FIGS. 6A, 6C, 6E, 6G, 6I and 6K are confocalmicroscope images of the eukaryotic cells containing gfp+ AMB-1; andFIGS. 6B, 6D, 6F, 6H, 6J, and 6L are fluorescence images of theeukaryotic cells containing gfp+ AMB-1.

FIGS. 7A-B show MDA-MB-231 cells containing lux+ AMB-1 imaged forluminescence or imaged with MRI. FIG. 7A is a chart of the luminescencesignal obtained from MDA-MB-231 cells with and without lux+ AMB-1. Unitsof the chart are luminescence per minute. FIG. 7B shows an MRI image ofMDA-MB-231 cells containing lux+ AMB-1 (dark band) where the MDA-MB-231cells containing lux+ AMB-1 are layered on top of an agarose base.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by way of example and not by way oflimitation. It should be noted that references to “an” or “one” or“some” embodiment(s) in this disclosure are not necessarily to the sameembodiment, and all such references mean at least one.

It is also to be understood that the singular terms “a”, “an”, and “the”include plural referents unless context clearly indicates otherwise.Similarly, the word “or” is intended to include “and” (and vice versa)unless the context clearly indicates otherwise. Numerical limitationsgiven with respect to concentrations or levels of a substance areintended to be approximate. Thus, where a concentration is indicated tobe at least (for example) 10 μg, it is intended that the concentrationbe understood to be at least approximately or about 10 μg.

In one aspect, the present invention is directed to eukaryotic cellscontaining single-celled organisms, such as host cells containingartificial endosymbionts in the cytosol of the host cell, and methods ofintroducing the single-celled organism into the eukaryotic cell. In oneembodiment, the single-celled organism is an artificial endosymbiontthat is genetically altered. In some embodiments, the single-celledorganisms are magnetotactic bacteria (MTB). The present invention isalso directed to eukaryotic cells engineered with a single-celledorganism to have multiple phenotypes for detection. Such multiplephenotypes for detection allow multimodal observation of the eukaryoticcells. In an embodiment, multimodal detection of the eukaryotic cells isused for non-invasive in vivo imaging. Each imaging method (or modality)allows the visualization of different aspects of anatomy and physiology,and combining these allows the imager to learn more about the target orsubject being imaged.

Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, the term “AMB” or “AMB-1” refers to Magnetospirillummagneticum strain AMB-1.

As used herein, the term “artificial endosymbiont” refers to asingle-celled organism which is or has been introduced into the cytosolof a eukaryotic cell through human intervention, and which has been orcan be transferred to daughter cells of the eukaryotic cell. In someembodiments, the single-celled organism maintains sufficient copy numberin the daughter cells so that a phenotype introduced by the artificialendosymbiont is maintained in the daughter cells.

As used herein, the term “cellular life cycle” refers to series ofevents involving the growth, replication, and division of a eukaryoticcell. Generally, it can be divided into five stages, known as G₀, inwhich the cell is quiescent, G₁ and G₂, in which the cell increases insize, S, in which the cell duplicates its DNA, and M, in which the cellundergoes mitosis and divides.

As used herein, the term “cytosol” refers to the portion of thecytoplasm not within membrane-bound sub-structures of the cell.

As used herein, the term “daughter cell” refers to cells that are formedby the division of a cell.

As used herein, the term “essential molecule” refers to a moleculeneeded by a cell for growth or survival.

As used herein, the term “genetically modified” refers to altering thegenetic material of a cell so that a desired property or characteristicof the cell is changed. The term includes introduction of heterologousgenetic material into the cell.

As used herein, the term “fluorescent protein” refers to a proteincapable of light emission when excited with an appropriateelectromagnetic radiation. Fluorescent proteins include proteins havingamino acid sequences that are either natural or engineered.

As used herein, the term “bioluminescent protein” refers to a form ofchemiluminescence which arises as the result of an energy-yieldingchemical reaction in which a specific biochemical substance, for examplea luciferin (a naturally occurring fluorophore), is oxidized by anenzyme (e.g., a luciferase) resulting in chemiluminescence.

As used herein, the term “host cell” refers to a eukaryotic cell inwhich an artificial endosymbiont can reside.

As used herein, the term “image modality” refers to absorption oremission of electromagnetic radiation, acoustic waves, nuclearparticles, or other types of energy (e.g., electrical), or a combinationthereof, which permits detection or interrogation of the system ortarget that contains it.

As used herein, the term “intracellular endosymbiont” refers tosingle-celled organism that spends at least part of its naturallife-cycle inside the cells of a eukaryotic organism.

As used herein, the terms “intracellular pathogen” and “intracellularparasite” refer to bacteria that infect a host organism, naturallycauses a disease in the host organism, and during the infection somebacteria enter host cells.

As used herein, the term “liposome mediated” refers to artificialvesicles having an aqueous core enclosed in one or more lipid layers,used to convey artificial endosymbionts to host cells.

As used herein, the term “luciferase” refers to a protein that uses achemical substrate to produce photons. In some embodiments, luciferaserefers to an enzyme or photoprotein, such as an oxygenase, thatcatalyzes a reaction that produces bioluminescence. Luciferases can berecombinant or naturally occurring, or a variant or mutant thereof.

As used herein, the term “magnetosome” refers to particles of a magneticmineral enclosed by a sheath or membrane, either as individual particlesor in chains of particles. In some embodiments, the magnetic mineral inthe magnetosome can comprise magnetite (i.e., Fe₃ O₄) or greigite(Fe₃S₄).

As used herein, the term “magnetic bacteria” refers to bacteria that areable to respond to an external magnetic field.

As used herein, the term “magnetotactic bacteria” or “MTB” refers tobacteria with genes encoding magnetosomes.

As used herein, the term “mammal” refers to warm-blooded vertebrateanimals all of which possess hair and, in the female, milk producingmammary glands.

As used herein, the term “microinjection” refers to the injection ofartificial endosymbionts into host cells.

As used herein, the term “parent cell” refers to a cell that divides toform two or more daughter cells.

As used herein, the term “phenotype” refers to the set of observablecharacteristics of an organism or cell.

As used herein, the term “receptor mediated” refers to a molecularstructure or site on the surface of a host cell that binds with abacterium or a tagged bacterium followed by internalization of thebacterium.

As used herein, the term “reporter” or “reporter molecule” refers to amoiety capable of being detected indirectly or directly. Reportersinclude, without limitation, a chromophore, a fluorophore, a fluorescentprotein, a receptor, a hapten, an enzyme, and a radioisotope.

As used herein, the term “reporter gene” refers to a polynucleotide thatencodes a reporter molecule that can be detected, either directly orindirectly. Exemplary reporter genes encode, among others, enzymes,fluorescent proteins, bioluminescent proteins, receptors, antigenicepitopes, and transporters.

As used herein, the term “reporter probe” refers to a molecule thatcontains a detectable label and is used to detect the presence (e.g.,expression) of a reporter molecule. The detectable label on the reporterprobe can be any detectable moiety, including, without limitation, anisotope (e.g., detectable by PET, SPECT, etc), chromophore, andfluorophore. The reporter probe can be any detectable molecule orcomposition that binds to or is acted upon by the reporter to permitdetection of the reporter molecule.

As used herein, the term “heterologous” when used in reference to anucleic acid or polypeptide refers to a nucleic acid or polypeptide notnormally present in nature. Accordingly, a heterologous nucleic acid orpolypeptide in reference to a host cell refers to a nucleic acid orpolypeptide not naturally present in the given host cell. For example, anucleic acid molecule containing a non-host nucleic acid encoding apolypeptide operably linked to a host nucleic acid comprising a promoteris considered to be a heterologous nucleic acid molecule. Conversely, aheterologous nucleic acid molecule can comprise an endogenous structuralgene operably linked with a non-host (exogenous) promoter. Similarly, apeptide or polypeptide encoded by a non-host nucleic acid molecule, oran endogenous polypeptide fused to a non-host polypeptide is aheterologous peptide or polypeptide.

As used herein, the term “secrete” refers to the passing of molecules orsignals from one side of a membrane to the other side.

As used herein, the term “selective agent” refers to a molecule, apolypeptide, or a set of culture conditions that are lethal orinhibitory to a single-celled organism, and/or an artificialendosymbionts, and/or host cells in the absence of a selectable agent.

As used herein, the term “tagged artificial endosymbiont” refers toartificial endosymbionts that have a ligand on the surface of theendosymbiont.

Artificial Endosymbionts

Single-celled organisms of the invention include bacteria that arecapable of surviving in a eukaryotic cell and maintain copy number suchthat the phenotype introduced by the single-celled organism is observedin daughter cells. In some embodiments, the single-celled organism doesnot kill the eukaryotic host cell without further human intervention. Insome embodiments, the single-cell organism has a functionality that isacquired by the eukaryotic cell following the introduction of thesingle-celled organism. In some embodiments, one or both of themodalities in multimodality provide additional functionality other thanjust imaging. In some embodiments, the functionality of the single-cellorganism is magnetism, production of a nutrient, metabolite, vitamin,cofactor, DNA molecule, RNA molecule, macromolecule, industrialprecursor, prodrug, hormone, fatty acid, carbohydrate, simple sugar,signal molecule, pharmacologically active compound, biologically activecompound, desalinization, cryoprotectant, nitrogen fixation,photosynthesis, response to environmental challenges, or tolerance toharsh environmental challenges. In some embodiments, the functionalityinvolves expression of a gene in the single-celled organism. In someembodiments, the functionality involves expression of one or more genesor set of genes in the single-celled organism. In some embodiments, thefunctionality involves expression of a protein in the single-celledorganism. In some embodiments the functionality involves expression of aset of proteins in the single-celled organism. In some embodiments, thefunctionality involves expression of a gene or gene product that istransferred to the host to express the phenotype.

Magnetism includes diamagnetism and paramagnetism. In some embodiments,magnetism includes ferromagnetism. In some embodiments, the eukaryoticcell maintains the functionality for at least 48 hours. In someembodiments, the single-celled organism can stably maintain phenotype inthe eukaryotic daughter cells through at least 2 cell divisions, or atleast 3 cell divisions, or at least 4 division, or at least 5 divisions,or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20cell divisions. In another embodiment, the single-celled organism canstably maintain phenotype in the eukaryotic daughter cells through 3-5divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 divisions.

In some embodiments, the single-celled organisms of the invention aregenetically modified. Methods for genetically modifying bacteria arewell known in the art. In some embodiments, the bacteria will begenetically modified to improve their survival in eukaryotic host cells,and/or to reduce the toxicity of the single-celled organism to theeukaryotic cell, and/or to provide the eukaryotic cell with a usefulphenotype. In one embodiment, the flagellar proteins of a single-celledorganism are modified so that the single-celled organism no longerexpresses flagellar proteins in the eukaryotic host cell. In anotherembodiment, the single-celled organism is modified so that it can nolonger synthesize an essential molecule that is preferably provided bythe eukaryotic host cell. In an embodiment, the single-celled organismis genetically modified so that its cell cycle is coordinated with thecell cycle of the eukaryotic host cell so that copy number of thesingle-celled organism can be maintained at a sufficient level to impartthe phenotype to daughter cells. In some embodiments, the genes localizeartificial endosymbionts to specific subcellular locations. In certainembodiments, the genes provide enhanced or blocked entry of theartificial endosymbionts to specific host cells. In some embodiments,the gene suppresses or alters the host immune system response to theartificial endosymbiont or genes and proteins expressed from it.

Embodiments of the invention include single-celled organisms that areα-Proteobacteria. In the current taxonomic scheme based on 16S rRNA,α-proteobacteria are recognized as a Class within the phylumProteobacteria, and are subdivided into 7 main subgroups or orders(Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales,Rickettsiales, Sphingomonadales and Parvularculales). (Gupta, R. S.,“Phylogenomics and signature proteins for the alpha Proteobacteria andits main groups,” BMC Microbiol. 7:106 (2007), incorporated herein byreference in its entirety for all purposes).

A large number of a-proteobacterial genomes that cover all of the maingroups within α-proteobacteria have been sequenced, providinginformation that can be used to identify unique sets of genes orproteins that are distinctive characteristics of various highertaxonomic groups (e.g., families, orders, etc.) within α-proteobacteria.(Gupta, supra; incorporated herein by reference in its entirety for allpurposes).

Single celled organisms useful as artificial endosymbionts include, byway of example and not limitation, Anabaena, Nostoc, Diazotroph,Cyanobacteria, Trichodesmium, Beijerinckia, Clostridium, Green sulfurbacteria, Azotobacteraceae, Rhizobia, Frankia, flavobacteria,Methanosarcinales, aerobic halophilic Archaea of the orderHalobacteriales, the fermentative anerobyves of the orderHalanaerobiales (low G+C brand of the Firmicutes), the red aerobicSalinibacter (Bacteroidetes branch), Marinobacter, Halomonas,Dermacoccus, Kocuria, Micromonospora, Streptomyces, Williamsia,Tskamurella, Alteromonas, Colwellia, Glaciecola, Pseudoalteromonas,Shewanella, Polaribacter, Pseudomonas, Psychrobacter, Athrobacter,Frigoribacterium, Subtercola, Microbacterium, Rhodoccu, Bacillus,Bacteroides, Propionibacterium, Fusobacterium, Klebsiella,lecithinase-positive Clostridia, Veillonella, Fusobacteria,Chromatiaceae, Chlorobiceae, Rhodospirillaceae, thiobacilli,nitrosomonas, nitrobacter, methanogens, acetogens, sulfate reducers, andlactic acid bacteria.

The genomes of a number of these single celled organisms have been orare being sequenced, including for example: M. frigidum, M. burtonii, C.symbiosum, C. psychrerythraea, P. haloplanktis, Halorubrumlacusprofundi, Vibrio salmonicida, Photobacterium profundum, S.violacea, S. frigidimarina, Psychrobacter sp. 273-4, S. benthica,Psychromonas sp. CNPT3, Moritella sp., Desulfotalea Psychrophila,Exiguobacterium 255-15, Flavobacterium psychrophilum, Psychroflexustorquis, Polaribacter filamentous, P. irgensii, Renibacteriumsalmoninarum, Leifsonia-related PHSC20-c1, Acidithiobacillusferrooxidans, Thermoplasma acidophilum, Picrophilus torridus, Sulfolobustokodaii, and Ferroplasma acidarmanus.

In an embodiment, artificial endosymbionts exclude single-celledorganisms that are known to be intracellular pathogens or intracellularendosymbionts. The genomes of many intracellular pathogens includegenomic islands containing virulence genes encoding, for example,adherence factors that allow the intracellular pathogen to attach totarget eukaryotic cells, and trigger phagocytosis of the intracellularpathogen. (Juhas, M. et al., “Genomic islands: tools of bacterialhorizontal gene transfer and evolution,” FEMS Microbiol Rev. 33:376-393(2009), incorporated herein by reference in its entirety for allpurposes). Many virulence factors utilize type III or type IV secretionsystems. Some virulence factors are secreted into the eukaryotic hostcell and alter membrane traffic within the target eukaryotic cell, somevirulence factors interact with host proteins involved in apoptosis.(Dubreuil, R. et al., “Bringing host-cell takeover by pathogenicbacteria to center stage,” Cell Logis. 1:120-124 (2011), incorporatedherein by reference in its entirety for all purposes).

Embodiments of the invention include single-celled organisms that aremagnetotactic bacteria (“MTB”). A large number of MTB species are knownto those of ordinary skill in the art since their initial discovery in1975 by Blakemore (see, e.g., Blakemore, R., “Magnetotactic bacteria,”Science 24: 377-379 (1975), incorporated herein by reference in itsentirety for all purposes) and represent a group of microbes (Faivre, D.et al., “Magnetotactic bacteria and magnetosomes,” Chem Rev.108:4875-4898 (2008), incorporated herein by reference in its entiretyfor all purposes). MTB have been identified in different subgroups ofthe Proteobacteria and the Nitrospira phylum with most of the phylotypesgrouping in α-Proteobacteria. Currently, culturable MTB strains assignedas α-Proteobacteria by 16S rRNA sequence similarity include the strainoriginally isolated by Blakemore in 1975, Magnetospirillummagnetotactium (formerly Aquasprillium magnetotactium), M.gryphiswaldense, M. magneticum strain AMB-1 (“AMB”), M. polymorphum,Magnetosprillum sp. MSM-4 and MSM-6, Magnetococcus marinus, marinevibrio strains MV-1 and MV-2, a marine spirillum strain MMS-1 andMagnetococcus sp. strain MC-1, as well as others. A number of MTB areavailable in pure culture, including AMB. The doubling time of AMB inpure culture is approximately eight hours and is close to that of atypical mammalian cell.

Standard MTB growth media uses succinic acid as the main carbon source,but MTB can be grown with fumarate, tartrate, malate, lactate, pyruvate,oxaloacetate, malonate, P-hydroxybutyrate and maleate as the sole carbonsource. These metabolites are present inside eukaryotic cells.Microaerophillic, facultative anaerobic, and obligate anaerobic MTBstrains have been identified. Oxygen concentrations in the cytosol ofeukaryotic cells are low due to sequestration by proteins such asmyoglobin and concentration in specific cellular locations, e.g.,mitochondria, thus the microaerophilic or facultative anaerobicenvironment necessary for MTB growth is already present in a eukaryoticcell.

MTBs can also be classified by the magnetic particles they synthesize,either magnetite (Fe₃O₄) or greigite (Fe₃S₄). Magnetite producers aremicroaerophilic or facultative anaerobic, need some oxygen source formagnetosome synthesis, and have optimal growth temperatures nearphysiological temperature.

In some embodiments, the single-celled organisms of the invention aregenetically modified. Molecular biology tools have been developed forgenetic manipulations of MTB most extensively in AMB and M.gryphiswaldense strain MSR-1 (reviewed in Jogler, C. and Schtiler, D.,in “Magnetoreception and Magnetosomes in Bacteria,” p 134-138, New York,Springer (2007), incorporated herein by reference in its entirety forall purposes). Because the genome of AMB was the first sequenced of anyMTB, all MTB gene references herein refer to this genome unlessotherwise specified. The genomes of two other Magnetospirillum strainsand Magnetococcus sp. strain MC-1 have also been recently sequenced.Genes from these strains or other MTB strains, presently culturable orunculturable, sequenced or unsequenced, known or unknown, can be used inthe present invention.

The genes responsible for magnetosome formation in MTB cluster ingenomic islands, known as the magnetosome island (MAI). In M.gryphiswaldense, the 130 kb MAI is generally structured into fourpolycistronic operons: the mamAB operon has 17 identified ORFs extendingover 16.4 kb; the mamGFDC operon has 4 identified ORFs, 2.1 kb and 15 kbupstream of mamAB; the mms6 operon has 6 identified ORFs, 3.6 kb and 368bp upstream of the mamGFDC; the mamXY operon has 4 identified ORFslocated about 30 kb downstream of mamAB; and the monocistronic mamWgene. In the Mal, the proteins Mam W, Mgl457, Mgl458, Mgl459, Mms6,Mgl462, MamG, MamF, MamD, MamC, MamH, MamI, MamE, MamJ, MamK, MamL,MamM, MamN, MamO, MamP, MamA, MamQ, MamR, MamB, MamS, MamT, MamU, andMgl505 have been identified, many of which have been given specificfunctions in magnetosome formation. Four genes outside the MAI have beenlinked to magnetosome formation, mamY, mtxA, mmsF and mamX. ConservedMAI's have been found in other MTB with some differences in genomicorganization and size. These genes have also been identified for AMB-1.(See Table 2 in Fukuda, Y. et al., “Dynamic analysis of a genomic islandin Magnetospirillum sp. Strain AMB-1 reveals how magnetosome synthesisdeveloped,” FEBS Lett. 580:801-812 (2006), incorporated herein byreference in its entirety for all purposes).

In some embodiments, genetic modifications are made to the single-celledorganism. Such modifications can be directed modifications, randommutagenesis, or a combination thereof. Natural endosymbionts are donorsof novel capabilities and often derive nutritional requirements from thehost.

Natural colonization of a host by the symbionts occurs in sevenstages: 1) transmission, 2) entry, 3) countering of host defense, 4)positioning, 5) providing advantage to the host, 6) surviving in hostenvironment, and 7) regulation.

In some embodiments, mutual nutritional dependence (biotrophy) may beestablished between the single-celled organism and the eukaryotic cell.In one embodiment, the single-celled organism comprises at least onedeletion or inactivation of a gene encoding an enzyme for synthesizingan essential molecule, thereby resulting in absence of enzyme orexpression of inactive enzyme, wherein said essential molecule isproduced by the eukaryotic host cell. An essential molecule can include,but is not limited to, an amino acid, a vitamin, a cofactor, and anucleotide. For instance, biotrophy can be accomplished by knocking-outthe ability of the single-celled organism to make an amino acid, whichwill then be derived from the host. Glycine is a reasonable choice as itis highly abundant in mammalian cells and a terminal product inbacterial amino acid biogenesis; at least 22 other possibilities exist.The enzyme serine hydroxymethyltransferase converts serine into glycineat the terminus of the 3-phosphoglycerate biosynthetic pathway for aminoacid production. In one embodiment, the single-celled organism is an AMBin which the gene amb2339 (which encodes the enzyme serinehydroxymethyltransferase) is genetically modified. There are numerousmethods for mutating or knocking-out genes known to those of ordinaryskill in the art, including in vitro mutagenesis, targeted insertion ofDNA into the gene of interest by homologous recombination or deletion ofthe gene (or operon, as most of the genes in the bacteria cluster inoperons), or using endonucleases provided appropriate sites only aroundthe target are present in the genome.

In another embodiment, nutritional dependence for a single-celledorganism on the host cell could also be established by eliminating theability of the single-celled organism to synthesize various metabolites,cofactors, vitamins, nucleotides, or other essential molecules.

In some embodiments of the invention, an MTB has mutations and/ordeletions in genes associated with mobility and/or secretion. MTB areflagellated, and in some embodiments of the invention, the MTB has adeletion and/or mutation in at least one gene encoding molecularmachinery associated with the flagella such that the magnetic bacteriumdoes not produce a functional flagellum. Additionally, many MTB secretevarious compounds, such as hydroxamate and catechol siderophores, whichmay be detrimental to or elicit an immune response from the host. In thesequenced genome of AMB, of the 4559 ORF's, 83 genes have been relatedto cell mobility and secretion. The flagellar assembly is known to becomposed of the gene products of amb0498, amb0500, amb0501, amb0502,amb0503, amb0504, amb0505, amb0610, amb0614, amb0615, amb0616, amb0617,amb0618, amb0619, amb0628, amb1289, amb1389, amb2558, amb2559, amb2578,amb2579, amb2856, amb3493, amb3494 amb3495, amb3496, amb3498, amb3824,and amb3827. The flagella is controlled by the chemotaxis machinery,which is composed of at least the gene products of amb0322, amb0323,amb0324, amb0325, amb0326, amb1806, amb1963, amb1966, amb2333, amb2635,amb2640, amb2648, amb2652, amb2826, amb2932, amb3002, amb3003, amb3004,amb3007, amb3102, amb3329, amb3501, amb3502, amb3654, amb3879, andamh3880.

In one embodiment, genes encoding antibiotic resistance are insertedinto the genome of the single-celled organism. Eukaryotic cells culturedin media containing the antibiotic will require the single-celledorganism for survival. Neomycin resistance is conferred by either one oftwo aminoglycoside phosphotransferase genes, which also provideresistance against geneticin (G418), a commonly used antibiotic foreukaryotes. Hygromycin B resistance is conferred by a kinase thatinactivates hygromycin B by phosphorylation. Puromycin is a commonlyused antibiotic for mammalian cell culture, and resistance is conferredby the pac gene encoding puromycin N-acetyl-transferase. Externalcontrol of the antibiotic concentration allows intracellular regulationof the copy number of the single-celled organism. Any other system whereresistance or tolerance to an external factor is achieved by chemicalmodification of this factor can also be employed. An indirect nutritiveadvantage on eukaryotic cells may also be established by using MTB and amagnetic culture method. In this embodiment, magnetic fields areestablished to confer an advantage to eukaryotic cells containing MTB.This could be either by providing the means for attachment to culturematrix, access to necessary growth or media factors or by selectionbetween cell passages.

In another embodiment, genetic modifications are made in the MTB genometo enhance intracellular stability against the host defense mechanismsfor a particular host cell type. Many eukaryote endosymbionts andendoparasites, such as the proteobacterial endosymbionts of insects suchas Buchnera, Wigglesivorthia, and Wolhachia; the methanogenicendosymbionts of anaerobic ciliates; the nitrogen-fixing symbionts inthe diatom Rhopalodia; the chemosynthetic endosymbiont consortia ofgutless tubeworms (Olavius or Jnanidrillus); the cyanobacterialendosymbionts of sponges; the endosymbionts of all five extant classesof Echinodermata; the Rhizobia endosymbionts of plants; variousendosymbiotic algae; the Legionella-like X bacteria endosymbionts ofAmeoba proteus; numerous Salmonella sp., Mycobacterium tuberculosis,Legionella pneumophila, etc. reside in membrane-bound vacuoles oftentermed symbiosomes, while some species, such as Blochmannia, therickettsia, Shigella, enteroinvasive Escherichia coli, and Listeria,have the ability to inhabit the cytosol. The Dot-Icm Type IV secretorysystem is employed by many intracellular bacteria acquired byphagocytosis to evade the endocytic pathway and persist in the hostcell. This system has been well-studied in L. pneumophila and consistsof the proteins: DotA through DotP, DotU, DotV, IcmF, IcmQ through IcmT,IcmV, IcmW and IcmX. In Photorhabdus luminescens, the luminescentendosymbiont of nematodes, the genes encoding RTX-like toxins,proteases, type III secretion system and iron uptake systems were shownto support intracellular stability and replication. The gene bacA andthe regulatory system BvrRS are essential for maintenance of symbiosisbetween Rhizobia and plants as well as the survival of Brucella ahortusin mammalian cells. The PrfA regulon enables some Listeria species toescape the phagesome and inhabit the cytosol. The desired cellularlocation (e.g., symbiosome or cytosol) of the intracellular MTB willdictate which genes are required to be expressed in the MTB (eitherdirectly from the genome or through a stable vector) for survival andproliferation in the host environment. The endogenous plasmid pMGT ishighly stable in MTB and a number of other broad range vectors(including those of IncQ, IncP, pBBRl, etc.) are capable of stablereplication in MTB.

In another embodiment, the single-celled organism is geneticallymodified by knocking in genes, such as bacteriostatic gene(s),siderophore gene(s), metabolic requirement gene(s), suicide gene(s),life cycle regulation gene(s), transporter gene(s), and escape from thephagosome gene(s). In another embodiment, the single-celled organismsare randomly mutated and subsequently screened for enhanced integrationwithin the host cell. Random mutation can be accomplished by treatmentwith mutagenic compounds, exposure to UV-light or other methods know tothose skilled in the art.

In another embodiment, transgenetic modification(s) are made to countereukaryotic cell defenses using genes from various parasites orendosymbionts. In another embodiment, the population of thesingle-celled organisms in the eukaryotic host cell is regulated thougha balance of intrinsic use of host mechanisms (nutrient availability,control of reproduction, etc.) and antibiotic concentration.

In another embodiment, a natural endosymbiont or an intracellularparasite is genetically modified to produce magnetosomes. Endosymbiontsof insects such as Buchnera, Wigglesworthia, and Wolbachia; themethanogenic endosymbionts of anaerobic ciliates; the nitrogen-fixingsymbionts in the diatom Rhopalodia; the chemosynthetic endosymbiontconsortia of gutless tubeworms (Olavius or Inanidrillus); thecyanobacterial endosymbionts of sponges; the endosymbionts of all fiveextant classes of Echinodermata; the Rhizobia endosymbionts of plants;various endosymbiotic algae; the Legionella-like X bacteriaendosymbionts of Ameoba proteus, numerous Salmonella sp., Mycobacteriumtuberculosis, Legionella pneumophila belonging to α-proteobacteria couldbe genetically engineered to produce magnetosomes. In anotherembodiment, a pre-existing organelle can be genetically modified toexpress one or more magnetosome genes to produce an artificialendosymbiont. For instance, mitochondria, plastids, hydrogenosomes,apicoplasts or other organelles, which harbor their own geneticmaterial, can be genetically altered. Bacteria modified to producemagnetosomes may include Francisella tularensis, Listeria monocytogenes,Salmonella typhi, Brucella, Legionella, Mycobacterium, Nocardia,Rhodococcus equi, Yersinia, Neisseria meningitidis, Chlamydia,Rickettsia, Coxiella and the like.

In a preferred embodiment, the single-celled organism is an MTB, whichmay or may not be genetically altered, that produces magnetic particlesupon culturing of the eukaryotic cells.

Nucleic Acids

The nucleic acids of the invention can include expression vectors, suchas plasmids, or viral vectors, or linear vectors, or vectors thatintegrate into chromosomal DNA. Expression vectors can contain a nucleicacid sequence that enables the vector to replicate in one or moreselected host cells. Such sequences are well known for a variety ofcells. The origin of replication from the plasmid pBR322 is suitable formost Gram-negative bacteria. In eukaryotic host cells, e.g., mammaliancells, the expression vector can be integrated into the host cellchromosome and then replicate with the host chromosome. Similarly,vectors can be integrated into the chromosome of prokaryotic cells.

Expression vectors also generally contain a selection gene, also termeda selectable marker. Selectable markers are well-known in the art forprokaryotic and eukaryotic cells, including host cells of the invention.This selection gene encodes a protein necessary for the survival orgrowth of transformed host cells grown in a selective culture medium.Host cells not transformed with the vector containing the selection genewill not survive in the culture medium. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline; (b)complement auxotrophic deficiencies; or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli. One example of a selection scheme utilizes a drugto arrest growth of a host cell. Those cells that are successfullytransformed with a heterologous gene produce a protein conferring drugresistance and thus survive the selection regimen. Other selectablemarkers for use in bacterial or eukaryotic (including mammalian) systemsare well-known in the art. In some embodiments, the selectable marker isthe target protein or encoded by the nucleic acid secreted by thesingle-celled organism into the host cell.

The expression vector for producing a heterologous polypeptide may alsocontain an inducible promoter that is recognized by the host RNApolymerase and is operably linked to the nucleic acid encoding thetarget protein. Inducible or constitutive promoters (or control regions)with suitable enhancers, introns, and other regulatory sequences arewell-known in the art.

In some embodiments, it may be desirable to modify the polypeptides ofthe invention. One of skill will recognize many ways of generatingalterations in a given nucleic acid construct. Such well-known methodsinclude site-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,e.g., Giliman and Smith, Gene 8:81-97 (1979) and Roberts et al., Nature328: 731-734 (1987), incorporated herein by reference.

In some embodiments, the recombinant nucleic acids encoding thepolypeptides of the invention are modified to provide preferred codonswhich enhance translation of the nucleic acid in a selected organism.

The polynucleotides of the invention also include polynucleotides,including nucleotide sequences that are substantially equivalent to thepolynucleotides of the invention. Polynucleotides according to theinvention can have at least about 80%, more typically at least about90%, and even more typically at least about 95%, sequence identity to apolynucleotide of the invention. The invention also provides thecomplement of the polynucleotides including a nucleotide sequence thathas at least about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotideencoding a polypeptide recited above. The polynucleotide can be DNA(genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithmsfor obtaining such polynucleotides are well known to those of skill inthe art and can include, for example, methods for determininghybridization conditions which can routinely isolate polynucleotides ofthe desired sequence identities.

Nucleic acids which encode protein analogs in accordance with thisinvention (i.e., wherein one or more amino acids are designed to differfrom the wild type polypeptide) may be produced using site directedmutagenesis or PCR amplification in which the primer(s) have the desiredpoint mutations. For a detailed description of suitable mutagenesistechniques, see Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989),and/or Ausubel et al., editors, Current Protocols in Molecular Biology,Green Publishers Inc. and Wiley and Sons, N.Y. (1994). Chemicalsynthesis using methods described by Engels et al., 1989, in Angew.Chem. Intl. Ed., Volume 28, pages 716-734, may also be used to preparesuch nucleic acids.

“Recombinant variant” refers to any polypeptide differing from naturallyoccurring polypeptides by amino acid insertions, deletions, andsubstitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added, or deletedwithout abolishing activities of interest, such as enzymatic or bindingactivities, may be found by comparing the sequence of the particularpolypeptide with that of homologous peptides and minimizing the numberof amino acid sequence changes made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides or chimeric polypeptides. Such alterations can, forexample, alter one or more of the biological functions or biochemicalcharacteristics of the polypeptides of the invention. For example, suchalterations may change polypeptide characteristics such asligand-binding affinities or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, or degradation/turnover rate.

In a preferred method, polynucleotides encoding the novel nucleic acidsare changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site of being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the art,and this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence is described inZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982).

PCR may also be used to create amino acid sequence variants of thenucleic acids. When small amounts of template DNA are used as startingmaterial, primer(s) that differs slightly in sequence from thecorresponding region in the template DNA can generate the desired aminoacid variant. PCR amplification results in a population of product DNAfragments that differ from the polynucleotide template encoding thetarget at the position specified by the primer. The product DNAfragments replace the corresponding region in the plasmid and this givesthe desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985), andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Ausubel et al.,supra.

Eukaryotic Cells

In some embodiments, the invention provides eukaryotic cells comprisingsingle-celled organisms in the eukaryotic cells that are heritable andmethods of introducing the single-celled organisms into host cells.

In some embodiments, the eukaryotic cells are plant cells. In someembodiments the eukaryotic cells are cells of monocotyledonous ordicotyledonous plants, including, but not limited to, maize, wheat,barley, rye, oat, rice, soybean, peanut, pea, lentil and alfalfa,cotton, rapeseed, canola, pepper, sunflower, potato, tobacco, tomato,eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass, ora forage crop. In other embodiments, the eukaryotic cells are algal,including but not limited to algae of the genera Chlorella,Chlamydomonas, Scenedesmus, Isochrysis, Dunaliella, Tetraselmis,Nannochloropsis, or Prototheca, In some embodiments, the eukaryoticcells are fungi cells, including, but not limited to, fungi of thegenera Saccharomyces, Klyuveromyces, Candida, Pichia, Debaromyces,Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.

In some embodiments, the eukaryotic cells of the invention are animalcells. In some embodiments the eukaryotic cells are mammalian, such asmouse, rat, rabbit, hamster, human, porcine, bovine, or canine. Miceroutinely function as a model for other mammals, most particularly forhumans. (See, e.g., Hanna, J. et al., “Treatment of sickle cell anemiamouse model with iPS cells generated from autologous skin,” Science318:1920-1923 (2007); Holtzman, D. M. et al., “Expression of humanapolipoprotein E reduces amyloid-β deposition in a mouse model ofAlzheimer's disease,” J Clin Invest. 103(6):R15-R21 (1999); Warren, R.S., et al., “Regulation by vascular endothelial growth factor of humancolon cancer tumorigenesis in a mouse model of experimental livermetastasis,” J Clin Invest. 95:1789-1797 (1995); each of which isincorporated herein by reference in its entirety for all purposes).

In some embodiments, the eukaryotic cell is a human cancer cell. Thereare many human cancer cell lines that are well known to those ofordinary skill in the art, including common epithelial tumor cell linessuch as Coco-2, MDA-MB-231 and MCF7; and non-epithelial tumor celllines, such as HT-1080 and HL60, and the NCI60-cell line panel (see,e.g., Shoemaker, R., “The NCI60 human tumor cell line anticancer drugscreen,” Nat Rev Cancer 6:813-823 (2006), incorporated herein byreference in its entirety for all purposes). Additionally, those ofordinary skill in the art are familiar with obtaining cancer cells fromprimary human tumors.

In other embodiments, the eukaryotic cells are stem cells. Those ofordinary skill in the art are familiar with a variety of stem celltypes, including Embryonic Stem Cells, Inducible Pluripotent Stem Cells,Hematopoietic Stem Cells, Neural Stem Cells, Epidermal Neural Crest StemCells, Mammary Stem Cells, Intestinal Stem Cells, Mesenchymal stemcells, Olfactory adult stem cells, and Testicular cells.

In an embodiment, the eukaryotic cell is a cell found in the circulatorysystem of a human host. For example, red blood cells, platelets, plasmacells, T-cells, natural killer cells, or the like, and precursor cellsof the same. As a group, these cells are defined to be circulating hostcells of the invention. The present invention may be used with any ofthese circulating cells. In an embodiment, the eukaryotic host cell is aT-cell. In another embodiment, the eukaryotic cell is a B-cell. In anembodiment, the eukaryotic cell is a neutrophil. In an embodiment, theeukaryotic cell is a megakaryocyte.

In another embodiment, at least one gene from the eukaryotic cell isgenetically altered. In some embodiments, mutual nutritional dependence(biotrophy) may be established between the artificial endosymbiont andthe eukaryotic cell by genetic modification of the eukaryotic cell,using the appropriate molecular biology techniques specific to thetarget host cell type known to those of ordinary skill in the art,creating eukaryotic cell dependence on the single-celled organism forsome essential macromolecule thus establishing the environmentalpressures for biotrophy. In another embodiment, nutritional dependencefor a single-celled organism on the eukaryotic cell may be establishedby genetically altering the single-celled organism to eliminate theability of it to synthesize various metabolites, cofactors, vitamins,nucleotides, or other essential molecules. In such embodiments, theessential molecule may be provided by the single-celled organism. Inanother embodiment, the eukaryotic cell gene encoding the enzyme serinehydroxymethyltransferase, which converts serine into glycine at theterminus of the 3-phosphoglycerate biosynthetic pathway for amino acidproduction, may be modified.

Methods of Introducing Single-Celled Organisms into Eukaryotic Cells

The single-celled organisms of the invention can be introduced intoeukaryotic cells by a number of methods known to those of skill in theart including, but not limited to, microinjection, natural phagocytosis,induced phagocytosis, macropinocytosis, other cellular uptake processes,liposome fusion, erythrocyte ghost fusion, electroporation, receptormediated methods, and the like (see, e.g., Microinjection and OrganelleTransplantation Techniques, Celis et al. Eds., Academic Press: New York,(1986), and references cited therein; incorporated herein by referencein its entirety for all purposes).

In one embodiment, a single-celled organism is introduced to the hostcell by microinjection into the cytoplasm of the host cell. A variety ofmicroinjection techniques are known to those skilled in the art.Microinjection is the most efficient of transfer techniques available(essentially 100%) and has no cell type restrictions (Id.; Xi, Z. etal., “Characterization of Wolbachia transfection efficiency by usingmicroinjection of embryonic cytoplasm and embryo homogenate,” ApplEnviron Microbiol. 71(6):3199-3204 (2005); Goetz, M. et al.,“Microinjection and growth of bacteria in the cytosol of mammalian hostcells,” Proc Natl Acad. Sci. USA 98:12221-12226 (2001); all publicationsincorporated herein by reference in its entirety for all purposes).

Naturally phagocytotic cells have been show to take up bacteria,including MTB (Burdette, D. L. et al., Vibrio VopQ induces PI3-kinaseindependent autophagy and antagonizes phagocytosis,” Mol Microbiol.73:639 (2009); Wiedemann, A. et al., “Yersinia enterocolitica invasintriggers phagocytosis via β1 integrins, CDC42Hs and WASp inmacrophages,” Cell Microbiol. 3:693 (2001); Hackam, D. J. et al., “Rhois required for the initiation of calcium signaling and phagocytosis byFcγ receptors in macrophages,” J Exp Med. 186(6):955-966 (1997);Matsunaga, T. et al., “Phagocytosis of bacterial magnetite byleucocytes,” Appl Microbiol Biotechnol. 31(4):401-405 (1989); allpublications incorporated herein by reference in its entirety for allpurposes).

This method is scalable, but may be limited to specific cell types(e.g., macrophage). However, recent studies have shown thatnon-phagocytotic cell types can be induced to endocytose bacteria whenco-cultured with various factors: media and chemical factors, andbiologic factors (e.g., baculovirus, protein factors, genetic knock-ins,etc.). (See, e.g., Salminen, M., et al., “Improvement in nuclear entryand transgene expression of baculoviruses by disintegration ofmicrotubules in human hepatocytes,” J Virol. 79(5):2720-2728 (2005);Modalsli, K. R. et al., “Microinjection of HEp-2 cells with coxsackie B1virus RNA enhances invasiveness of Shigella flexneri only afterprestimulation with UV-inactivated virus,” APMIS 101:602-606 (1993);Hayward, R. D. et al., “Direct nucleation and bundling of actin by theSipC protein of invasive Salmonella,” EMBO J. 18:4926-4934 (1999);Yoshida, S. et al., “Shigella deliver an effector protein to triggerhost microtubule destabilization, which promotes RacI activity andefficient bacterial internalization,” EMBO J. 21:2923-2935 (2002);Bigildeev et al. J Exp Hematol. 39:187 (2011); Finlay, B. B. et al.,Common themes in microbial pathogenicity revisited,” Microbiol Mot BlotRev. 61:136-169 (1997); all publications are incorporated herein byreference in its entirety for all purposes).

The related process, macropinocytosis or “cell drinking,” is a methodnumerous bacteria and viruses employ for intracellular entry (Zhang(2004) In: Molecular Imaging and Contrast Agent Database (MICAD)(database online); Bethesda (Md.): National Library of Medicine (US),NCBI; 2004-2011; both publications incorporated by reference in itsentirety for all purposes). Various protocols exist which can beemployed to induce cells to take up bacteria. Several agents, such asnucleic acids, proteins, drugs and organelles have been encapsulated inliposomes and delivered to cells (Ben-Haim, N. et al., “Cell-specificintegration of artificial organelles based on functionalized polymervesicles,” Nano Lett. 8(5):1368-1373 (2008); Lian, W. et al.,“Intracellular delivery can be achieved by bombarding cells or tissueswith accelerated molecules or bacteria without the need for carrierparticles,” Exp Cell Res. 313(1):53-64 (2007); Heng, B. C. et al.,“Immunoliposome-mediated delivery of neomycin phosphotransferase for thelineage-specific selection of differentiated/committed stem cellprogenies: Potential advantages over transfection with marker genes,fluorescence-activated and magnetic affinity cell-sorting,” MedHypotheses 65(2):334-336 (2005); Potrykus, Ciba Found Symp, Vol. 154:198 (1990); all publications incorporated herein by reference in itsentirety for all purposes). This method is inexpensive, relativelysimple and scalable. Additionally, liposome uptake can be enhanced bymanipulation of incubation conditions, variation of liposome charge,receptor mediation, and magnetic enhancement. (See, e.g., Pan et al. IntJ Pharm. 358:263 (2008); Sarbolouki, M. N. et al., “Storage stability ofstabilized MLV and REV liposomes containing sodium methotrexate (aqueous& lyophilized),” J Pharm Sci Techno. 52(10):23-27 (1998); Elorza, B., etal., “Comparison of particle size and encapsulation parameters of threeliposomal preparations,” J Microencapsul. 10(2):237-248 (1993);Mykhaylyk, O. et al., “Liposomal Magnetofection,” Methods Mol Bio.605:487-525 (2010); all publications incorporated herein by reference inits entirety for all purposes).

Erythrocyte-mediated transfer is similar to liposome fusion and has beenshown to have high efficiency and efficacy across all cell types tested(Microinjection and Organelle Transplantation Techniques, Celis et al.Eds.; Academic Press: New York (1986), incorporated by reference in itsentirety for all purposes). Typically erythrocytes are loaded by osmoticshock methods or electroporation methods (Schoen, P. et al., :Genetransfer mediated by fusion protein hemagglutinin reconstituted incationic lipid vesicles,” Gene Ther. 6:823-832 (1999); Li, L. H. et al.,“Electrofusion between heterogeneous-sized mammalian cells in a pellet:potential applications in drug delivery and hybridoma formation,”Biophys J. 71:479-486 (1996); Carruthers, A. et al., “A rapid method ofreconstituting human erythrocyte sugar transport proteins,” Biochem.23:2712-2718 (1984); each publication is incorporated herein byreference in its entirety for all purposes). Alternatively, erythrocytesmay be loaded indirectly by loading hematopoietic progenitors withsingle-celled organisms and inducing them to differentiate and expandinto erythrocytes containing single-celled organisms.

Electroporation is a commonly used, inexpensive method to deliverfactors to cells. (Potrykus, I., “Gene transfer methods for plants andcell cultures,” Ciba Found Symp. 154:198-208, discussion 208-12 (1990);Wolbank, S. et al., “Labeling of human adipose-derived stem cells fornon-invasive in vivo cell tracking,” Cell Tissue Bank 8:163-177 (2007);each publication incorporated herein by reference in its entirety forall purposes).

In another embodiment, a eukaryotic cell that naturally endocytosesbacteria (e.g., Chinese hamster ovary (CHO)) is used. In one embodiment,the modified single-celled bacteria are added to the CHO culturedirectly. CHO cells are cultured by standard procedures, for example inHam's F-12 media with 10% fetal calf serum media, prior to infectionwith the MTB. Post infection, the media is augmented with additionaliron (40 to 80 μM) as either ferric malate or FeCl₃. Numerous other celltypes internalize bacteria by endocytosis or more specificallyphagocytosis; endosymbionts or parasites have their own methods forcellular entry and these natural processes can be exploited forinternalization of the artificial endosymbionts resulting in thegeneration of so-called symbiosomes. In another embodiment, symbiosomesfrom one cell can be transplanted to another cell type (i.e., oneincapable of endocytosis of artificial endosymbionts) usingmicroinjection, organelle transplantation, and chimera techniques. Thesehost cells are cultured in typical media and with the techniques for thespecific cell type.

In one embodiment, a single-celled organism is introduced to the hostcell by a liposome mediated process. Mitochondria and chloroplasts,which are larger than MTB, have been efficiently introduced intoeukaryotic cells when encapsulated into liposomes (Bonnett, H. T. Planta131:229 (1976); Giles, K. et al., “Liposome-mediated uptake ofchloroplasts by plant protoplasts,” In Vitro Cellular & DevelopmentalBiology—Plant 16(7):581-584 (1980); each publication incorporated hereinby reference in its entirety for all purposes). Numerous liposome fusionprotocols and agents are available and can be used by the skilledartisan without undue experimentation (see, e.g., Ben-Haim, N. et al.,“Cell-specific integration of artificial organelles based onfunctionalized polymer vesicles,” Nano Lett. 8(5):1368-1373 (2008);Lian, W. et al., “Intracellular delivery can be achieved by bombardingcells or tissues with accelerated molecules or bacteria without the needfor carrier particles,” Exp Cell Res. 313(1):53-64 (2007); Heng, B. C.et al., “Immunoliposome-mediated delivery of neomycin phosphotransferasefor the lineage-specific selection of differentiated/committed stem cellprogenies: Potential advantages over transfection with marker genes,fluorescence-activated and magnetic affinity cell-sorting,” MedHypotheses 65(2):334-336 (2005); Potrykus, Ciba Found Symp, 1(54):198(1990); each publications incorporated herein by reference in itsentirety for all purposes).

Methods of Use of Eukaryotic Cells Comprising Single-Celled Organisms

In another aspect, the invention provides methods of using phenotypesintroduced into eukaryotic cells by single-celled organisms of theinvention. In some embodiments, the phenotype used is a heritablefunctionality not otherwise present in the eukaryotic cells. In someembodiments, eukaryotic cells with a magnetic phenotype are magneticallymanipulated.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes can be detected and monitored using magnetic detection orimaging techniques such as magnetic resonance imaging (MRI), magneticparticle imaging (MPI), magnetic relaxation switching (MRS), magneticresonance, superconducting quantum interference (SQUID), magnetometers,nuclear magnetic resonance (NMR), Mossbauer spectrometers, electronparamagnetic resonance (EPR), and magnetic circular dichroism. MRI is awidely used clinical diagnostic tool because it is non-invasive, allowsviews into optically opaque subjects (including mice, humans, and othermammals), and provides contrast among soft tissues at reasonably highspatial resolution, compared to non-magnetic imaging (such as opticalprobes) which tend to have low special resolution and to be limited inpenetration depth. MPI is a diagnostic method, which like MRI, isnon-invasive; however it specifically detects the magnetic fieldsgenerated by superparamagnetic iron oxide nanoparticles and results inimages with a very low background. Conventional MRI focuses almostexclusively on visualizing anatomy and has no specificity for anyparticular cell type. The ‘probe’ used by conventional MRI is theubiquitous proton ¹H in mobile water molecules. Contrast agents can beused for cell-type specificity, but contrast agents dilute or havetoxicology issues, and can only be used for short-term studies. Someembodiments of this invention facilitate cell-specific MRI, MPI or othermagnetic detection imaging in living subjects for longer-term studies.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are mammalian cancer cells, including human cancer celllines, for example human cancer cell line NCI 60; murine cancer celllines; and canine cancer cell lines. These magnetic cancer cells can beinjected into immunocompromised mammals such as mice and can then bemonitored with magnetic imaging to track tumor progression over time. Insome embodiments, anti-cancer treatments or putative treatments may beprovided to the immunocompromised mammal during the period that tumorprogression is being tracked in real time. In some embodiments,viability of eukaryotic cells of the invention with magnetic phenotypesis monitored using MRI, MPI or other magnetic detection means to assessin vivo cell response to different conditions, including drugtreatments.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are metastatic cancer cells that are introduced intoexperimental animals by methods including injection. MRI, MPI or othermagnetic detection can then be used to monitor the process of metastasisand movement of metastatic cancer cells throughout the experimentalanimals. In some embodiments magnetic eukaryotic cancer cells of theinvention are injected into a tumor bearing mammal, such as a mouse, andMRI, MPI or other magnetic detection is used to track metastatic cellcirculation through the mammal.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are macrophages and are injected into experimental animals.Magnetic imaging is used to detect any aggregations of macrophageswithin the animals. Macrophages aggregate to the sites of inflammation,which can be caused by malignant lesions including metastasis.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are stem cells or were derived from stem cells, including EScells, iPS cells, or adult stem cells obtained from mammalian species,including but not limited to, human, mouse, rat, and pig. Stem cells maybe introduced into a target organism directly or may be firstdifferentiated in vitro and then introduced into a target organism. Thein vivo fate, including localization, growth rates and viability, of theintroduced cells can be assayed through magnetic imaging.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are therapeutic T-cells. Therapeutic T-cells may beintroduced into a target organism directly, and the in vivo fate,including localization, growth rates and viability, of the introducedcells can be assayed through magnetic imaging.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are hematopoietic stem or progenitor cells, which are thenintroduced into a mammal. When hematopoietic stem or progenitor cellsare introduced into mammals, these cells will reside in the bone marrow.The behavior of the magnetic hematopoietic stem or progenitor cells,including their localization, proliferation and mobilization into bloodstream upon receiving different stimuli, can be monitored throughmagnetic imaging.

In some embodiments, magnetic artificial endosymbionts divide moreslowly than stem cell host cells in which they reside. Over time, stemcells, which generally divide more slowly than more differentiatedprogenitor cells, will retain magnetic phenotype longer than moredifferentiated progenitor cells and the two types of cells will becomemeasurably distinct when imaged magnetically. In some embodiments,eukaryotic cells of the invention with magnetic phenotypes are fused toa eukaryotic cell line of a desired cell type, creating chimeric cells.Chimeric cells can be introduced into an animal and tracked by magneticimaging.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are embryonic cells. In some embodiments, the embryonic cellsare fertilized animal eggs, such as mouse or rat. In some embodiments,embryos are implanted into female animals and allowed to develop,leading to the production of animals with cells containing single-celledorganism throughout their bodies. Magnetic tissues can be harvested fromthe resulting organisms and magnetic cell lines can be derived fromthem. In some embodiments, these animals are bred and the magneticphenotype is inherited maternally. In some embodiments, eukaryotic cellsof the invention with magnetic phenotypes are introduced intomulti-celled embryos. The cell lineage of the magnetic cell can betracked by magnetic imaging as the embryo develops. In some embodiments,the artificial endosymbiont is not retained in the adult animal, but bytheir presence in the early stages of development the immune system ofthis animal does not recognize artificial endosymbionts or host cellswith artificial endosymbionts as foreign.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are moved by magnetically attracting the eukaryotic cells. Insome embodiments, this movement is achieved using externally generatedmagnetic fields and field gradients. Various devices have been reportedfor magnetic targeting, such as those in U.S. Pat. No. 8,159,224 andRiegler J. et al., “Superparamagnetic iron oxide nanoparticle targetingof MSCs in vascular injury,” Biomaterials 34(8):1987-94 (2013). In someembodiments, eukaryotic cells of the invention with magnetic phenotypesare separated from a heterogeneous population of non-magnetic cells,either in vitro or in vivo (following introduction into an organism) byusing a magnet to attract the magnetic cells.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are introduced into the bloodstream or other fluids of atarget organism. The eukaryotic cells can be directed to an area ofinterest on the organism and localized there with an aid of a magnetpositioned adjacent to this area. In some embodiments, the eukaryoticcells can be stem cells that are directed to and held in an area on amammal's body where they could have therapeutic effect. In someembodiments, the eukaryotic cells can be immune cells which can bedirected to a particular location on a mammal's body, including to atumor or injury site. In some embodiments, the eukaryotic cells can beloaded with a therapeutic agent that can be released after beingmagnetically directed to a desired area.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are placed in an alternating magnetic field, or alternativemagnetic field, referred to as AMF, for a technique called MagneticHyperthermia Technique (MHT). AMF and MHT are described in U.S.Publication No. US20120302819 (U.S. application Ser. No. 13/510,416);and in Silva A. C. et al., “Application of hyperthermia induced bysuperparamagnetic iron oxide nanoparticles in glioma treatment,” Int JNanomedicine 6:591-603 (2011); each publication incorporated herein byreference in its entirety for all purposes). Hyperthermia is atherapeutic procedure that promotes the increase of temperature in bodytissues in order to change the functionality of the cellular structures.Its activity is based on the fact that a temperature increase can inducecell damage, including cell lysing and cell death. In some embodiments,the eukaryotic cells of the invention are subjected to an alternatingmagnetic field for 1, 5, 10, 20, 30, 40, 50 or 60 minutes. In someembodiments, magnetic field frequencies of an applied AMF lie between 50kHz and 1 MHz. In some embodiments, the magnetic field amplitude of anapplied AMF remains below 100 mT. In some embodiments, the eukaryoticcells of the invention subjected to MHT are tumor cells, which are lessresistant to sudden increases in temperature than the normal surroundingcells. In some embodiments, the eukaryotic cells of the invention aretumor cells or are next to tumor cells and are subjected to analternating magnetic field until the internal temperature of the tumorreached between 43° C. and 47° C.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are placed in a spinning magnetic field, resulting inrotation of the single-celled organisms inside the cells and celldamage, including cell death. In some embodiments, eukaryotic cells ofthe invention with magnetic phenotypes in a heterogeneous population ofnon-magnetic cells are selectively damaged by subjecting the entire cellpopulation to an alternating magnetic field or to a spinning magneticfield. In some embodiments, eukaryotic cells of the invention withmagnetic phenotypes in a heterogeneous population of non-magnetic cellsare placed in an alternating magnetic field or a spinning magneticfield, resulting in damage to both the eukaryotic cells of the inventionand the non-magnetic cells located near the cells of the invention. Insome embodiments, eukaryotic cells of the invention with magneticphenotypes are introduced into an animal and are targeted to a locationwithin the animal by magnetic manipulation or other forms of celltargeting known in the art. The location within the animal can then besubjected to an alternating magnetic field or a spinning magnetic field,resulting in the damage to cells surrounding the magnetic cells. In someembodiments, eukaryotic cells of the invention with magnetic phenotypesare introduced into an animal and are targeted to a tumor site withinthe animal by magnetic manipulation or other forms of cell targetingknown in the art. The tumor can then be subjected to an alternatingmagnetic field or a spinning magnetic field, resulting in the damage totumor cells surrounding the magnetic cells. In some embodiments,eukaryotic cells of the invention with magnetic phenotypes are stemcells, including ES cells, iPS cells, or adult stem cells obtained frommammalian species including but not limited to human, mouse, rat, andpig. Stem cells may be introduced into a target organism directly or maybe first differentiated in vitro and then introduced into a targetorganism. Following introduction, the animal can be subjected to analternating magnetic field or a spinning magnetic field, resulting inthe death of introduced stem cells and resulting lineages of these stemcells. The in vivo fate, including localization, growth rates andviability, of the introduced cells can be assayed through magneticimaging.

Multimodal Detection

In another aspect, the present invention is directed to eukaryotic cellsengineered with a single-celled organism, such as an artificialendosymbiont. The singled-celled organism may impart at least onedesired phenotype into the eukaryotic cell. In some embodiments, thesingle-celled organism may secrete to and/or transport from the hostcell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), aminoacid(s), or other factor(s). This communication between the singlecelled organism and the host cell may result in the desired phenotype orphenotypes for the eukaryotic cell. Such a desired phenotype or desiredphenotypes may allow for multimodal detection or observation of theeukaryotic cells. Thus, in some embodiments, the single cell organismcan be used as multimodal probes for multimodal detection or observationof the eukaryotic cells.

In some embodiments, the multimodal probe comprises a magnetic bacteriaexpressing one or more reporters for multimodal detection. In someembodiments, the magnetic bacteria comprise a magnetotactic bacteriaexpressing one or more reporters for multimodal detection. In someembodiments, for use as a multimodal probe, a heterologous gene encodingthe reporter protein is introduced into the magnetic bacteria such thatthe genetically modified magnetic bacteria express the reporter. In someembodiments, the magnetic bacteria are engineered to express a singlereporter. In some embodiments, different magnetic bacteria, eachexpressing a different reporter, is introduced into a eukaryotic cellfor multimodal detection. In some embodiments, the magnetic bacteria isengineered to express two or more reporter products, for example byusing a single vector construct encoding two or more reporters. In someembodiments, the reporters comprise heterologous reporters and areexpressed in a form functional for detection. The expression of reportergenes provides for additional imaging modalities, in addition to themagnetic phenotype of the magnetic bacteria.

For example, the present disclosure shows effective reporter geneexpression for two modalities using the magnetotactic bacterial strainMagnetospirillum magneticum (AMB-1). In some embodiments for expressinga reporter gene, a plasmid containing a reporter gene and a suitableresistance gene (e.g., one which confers antibiotic resistance) can beintroduced into Escherichia coli cells, mating the E. coli cells withthe target magnetotactic bacterial cells, and then performing selectionon the magnetotactic bacteria. An appropriate assay can then beperformed on the transformed magnetotactic bacteria to confirm thepresence and positive expression of the reporter. In some embodiments,when selecting a reporter gene for incorporation, care is taken toensure that it can still be expressed in a functional form usingprokaryotic transcriptional and translational machinery. These modifiedbacteria have a magnetic phenotype and the phenotype of the reportergene, which allow for multimodal detection of eukaryotic cells carryingthe genetically modified magnetotactic bacteria. Introduction andexpression of additional reporter genes, where each reporter gene isdetectable using a different imaging modality, provides for additionalimaging capabilities.

In some embodiments, the magnetosomes of the genetically modifiedmagnetic bacteria can be detected using MRI systems, magnetic particleimaging (MPI) systems, magnetic relaxation switching (MRS) systems,magnetic resonance spectrometers, superconducting quantum interferencedevices (SQUID), magnetometers, nuclear magnetic resonance (NMR)systems, Mossbauer spectrometers, electron paramagnetic resonance (EPR)systems, and magnetic circular dichroism systems. The product of thereporter gene can be detected by any appropriate detection method andapparatus, depending on the type of reporter product expressed from thereporter gene. By way of example, an exemplary reporter gene encodes alight producing protein (e.g., luciferase or eGFP), and this phenotypecan be detected using optical imaging, which can be performed eithernon-invasively (for BLI and FLI) or with some degree of invasiveness,for example, intravital microscopy and fluorescence laparoscopy (see.e.g., Gahlen, J. et al., “Laparoscopic fluorescence diagnosis forintraabdominal fluorescence targeting of peritoneal carcinosis,” AnnSurg. 235:252-260 (2002), incorporated herein by reference in itsentirety for all purposes). In the descriptions herein, expression of areporter is meant to include expression of the corresponding reportergene resulting in expression of the encoded reporter or reportermolecule.

In some embodiments, the multimodal probe comprises a magnetic (e.g.,magnetotactic) bacterial cell expressing one or more reporters selectedfrom a Positron Emission Tomography (PET) reporter, a Single PhotonEmission Computed Tomography (SPECT) reporter, an X-Ray reporter, aphotoacoustic reporter, and an ultrasound reporter.

In some embodiments, the multimodal probe further expresses, in additionto the above reporters, a fluorescent reporter or a bioluminescentreporter. In some embodiments, the multimodal probe further expresses,in addition to the above reporters, a fluorescent and a bioluminescentreporter.

In some embodiments, the multimodal probe expresses at least a PositronEmission Tomography (PET) reporter. Various PET reporters can beexpressed and used in the multimodal probes. In some embodiments, thePET reporter comprises a thymidine kinase. In some embodiments, thethymidine kinase is selected from a Herpes Simplex Virus thymidinekinase, Varicella-Zoster Virus thymidine kinase, human mitochondrialthymidine kinase or active variants thereof (see, e.g., Campbell et al.,J Biol Chem. 287(1):446-54 (2012)). PET detection of thymidine kinase isgenerally achieved by using a PET-specific reporter probe. Exemplary PETreporter probe for HSV thymidine kinase includes[¹⁸F]9-(4-[18F]-fluoro-3-hydroxymethylbutyl)-guanine, afluorine-18-labelled penciclovir analogue, which when phosphorylated bythymidine kinase (TK) becomes retained intracellularly. Anotherthymidine kinase reporter probe is 5-(76)Br-bromo-2′-fluoro-2′-deoxyuridine. In some embodiments, a thymidinekinase reporter probe that is preferentially acted on by theheterologous thymidine kinase as compared to any endogenous thymidinekinase is used.

Other PET reporters that can be used in the multimodal probe include,among others, dopamine D2 (D2R) receptor, sodium iodide transporter(NIS), dexoycytidine kinase, somatostatin receptor subtype 2,norepinephrine transporter (NET), cannaboid receptor, glucosetransporter (Glut1), tyrosinase, and active variants thereof. Therelevant reporter probes for each of the PET reporters are well known tothe skilled artisan. An exemplary reporter probe for dopamine D2 (D2R)receptor is 3-(2′-[¹⁸F]fluoroethyl)spiperone (FESP) (MacLaren et al.,Gene Ther. 6(5):785-91 (1999)). An exemplary reporter probe for thesodium iodide transporter is ¹²⁴I, which is retained in cells followingtransport by the transporter. An exemplary reporter probe fordeoxycytidine kinase is2′-deoxy-2′-¹⁸F-5-ethyl-1-β-d-arabinofuranosyluracil (¹⁸F-FEAU). Anexemplary reporter probe for somatostatin receptor subtype 2 is ¹¹In—,^(99m)/^(94m)Tc—, ⁹⁰Y—, or ¹⁷⁷Lu-labeled octreotide analogues, forexample ⁹⁰Y—, or ¹⁷⁷Lu-labeled DOTATOC (Zhang et al., J Nucl Med.50(suppl 2):323 (2009)); ⁶⁸Ga-DOTATATE; and ¹¹¹In-DOTABASS (see. e.g.,Brader et al., J Nucl Med. 54(2):167-172 (2013), incorporated herein byreference). An exemplary reporter probe for norepinephrine transporteris ¹¹C-m-hydroxyephedrine (Buursma et al., J Nucl Med. 46:2068-2075(2005)). An exemplary reporter probe for the cannaboid receptor is¹¹C-labeled CB₂ ligand, ¹¹C-GW405833 (Vandeputte et al., J Nucl Med.52(7):1102-1109 (2011)). An exemplary reporter probe for the glucosetransporter is [¹⁸F]fluoro-2-deoxy-d-glucose (Herschman, H. R., Crit RevOncology/Hematology 51:191-204 (2004)). An exemplary reporter probe fortyrosinase is N-(2-(diethylamino)ethyl)-¹⁸F-5-fluoropicolinamide (Qin etal., Sci Rep. 3:1490 (2013)). Other reporter probes are described in theart, for example, in Yaghoubi et al., Theranostics 2(4):374-391 (2012),incorporated herein by reference.

In some embodiments, the multimodal probe expresses at least a SinglePhoton Emission Computed Tomography (SPECT) reporter. Exemplary SPECTreporters include sodium iodide transporter, dopamine D2 (D2R) receptor,and active variants thereof. In some embodiments, the SPECT reportercomprises a modified haloalkane dehalogenase capable of covalentlybonding a SPECT detectable chloroalkane substrate (see, e.g., Hong etal., Am J Transl Res. 5(3):291-302 (2013), incorporated herein byreference).

In some embodiments, the multimodal probe expresses at least aphotoacoustic reporter. Exemplary photoacoustic reporters include, amongothers, tyrosinase and β-galactosidase (see, e.g., Krumholz et al., JBiomed Optics. 16(8):1-3 (2011)). Reporter probes for tyrosinase havebeen described above. An exemplary reporter probe for β-galactosidase is5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (Li et al., J BiomedOpt. 12(2):020504 (2007)).

In some embodiments, the multimodal probe expresses at least an X-rayreporter. Exemplary X-ray reporter includes, among others, somatostatinreceptor 2, or other types of receptor based binding agents. Thereporter probe can have a radiopaque label moiety that is bound to thereporter probe and imaged, for example, by X-ray or computer tomography.Exemplary radiopaque label is iodine, particularly a polyiodinatedchemical group (see, e.g., U.S. Pat. No. 5,141,739), and paramagneticlabels (e.g., gadolinium), which can be attached to the reporter probeby conventional means.

In some embodiments, the multimodal probe expresses at least anultrasound reporter. Exemplary ultrasound reporter includes, amongothers, a binding agent that is capable of binding an ultrasoundcontrast agent, for example, microbubble contrast agent. For example,the binding agent can comprise an antibody expressed in themagnetotactic bacteria and directed specifically against a peptide,where the peptide is bound to microbubble contrast agents (see, e.g.,Kiessling et al., J Nucl. Med. 53:345-348 (2012)).

In the embodiments herein where the reporter is a fluorescent reporter,any number of fluorescent reporters can be used. These include, forexample, green fluorescent protein from Aequorea victoria or Renillareniformis, and active variants thereof (e.g., blue fluorescent protein,yellow fluorescent protein, cyan fluorescent protein, etc.); fluorescentproteins from Hydroid jellyfishes, Copepod, Ctenophora, Anthrozoas, andEntacmaea quadricolor, and active variants thereof; andphycobiliproteins and active variants thereof.

In some embodiments herein where the reporter is a bioluminescentreporter, any number of bioluminescent proteins can be used as thereporter. These include, by way of example and not limitation, aequorin(and other Ca⁺² regulated photoproteins), luciferase based on luciferinsubstrate, luciferase based on Coelenterazine substrate (e.g., Renilla,Gaussia, and Metridina), and luciferase from Cypridina, and activevariants thereof.

In some embodiments, the multimodal probe expresses at least two or morereporters, at least three or more reporters, at least four or morereporters, or at least five or more reporters selected from afluorescent reporter, a bioluminescent reporter, a PET reporter, a SPECTreporter, an X-ray reporter, a photoacoustic reporter, and ultrasoundreporter.

In some embodiments, the multimodal probe expresses at least tworeporters selected from a fluorescent reporter, a bioluminescentreporter, a PET reporter, a SPECT reporter, an X-ray reporter, aphotoacoustic reporter, and ultrasound reporter.

In some embodiments, the multimodal probe expresses at least threereporters selected from a fluorescent reporter, a bioluminescentreporter, a PET reporter, a SPECT reporter, an X-ray reporter, aphotoacoustic reporter, and ultrasound reporter.

In some embodiments, the reporter gene for the multimodal probe encodesa reporter that is detectable by multiple imaging modalities, forexample tyrosinase which has been shown to yield photoacoustic imaging(PAI), MRI and PET (with a suitable radiotracer) signals (see, e.g.,Qin, C. et al., “Tyrosinase as a multifunctional reporter gene forphotoacoustic/MRI/PET triple modality molecular imaging,” ScientificRep. 3:1490 (2013), incorporated herein by reference in its entirety forall purposes). Alternatively, the reporter gene can be a fusion proteincomprising two or more reporters linked together (e.g., aluciferase-GFP-thymidine kinase triple fusion reporter). (Ray P. et al.,“Imaging tri-fusion multimodality reported gene expression in livingsubjects,” Cancer Res. 64:1323-1330 (2004), incorporated herein byreference in its entirety for all purposes). In some embodiments, eachof the reporters comprises different reporters, each reporter beingdetectable by a different imaging modality.

In some embodiments, the gene expressing the heterologous reporter canbe extrachromosomal, for example, as part of self-replicating plasmids,cosmids, or bacmids. In some embodiments, the genes expressing thereporter can be integrated into the bacterial chromosome, for examplethrough homologous recombination, or integration mediated byrecombinases, such a Cre-loxP, Dre-rox, yeast flippase (FLP), and attTn7based integration (Choi et al., Appl Environ Microbiol. 72(1):753-758(2008)). In some embodiments, the reporter gene is constructed toexpress the functional reporter without fusions to other proteins. Insome embodiments, the reporter gene is constructed to express thefunctional reporter as fusions, i.e., a fusion protein, for example toother heterologous proteins or proteins of the magnetotactic bacteria.

In some embodiments, genetically modified magnetic bacteria areintroduced to eukaryotic cells for multimodal detection. Once inside theeukaryotic cell, the genetically modified magnetic bacteria have thecapacity to self-replicate, thus maintaining their levels even after acell division event, which makes them a powerful tool for long term celltracking and other imaging capabilities. The magnetotactic bacteria'smagnetite-based iron cores are capable of generating a powerful in vivoMRI signal. Moreover, the genetically modified magnetic bacteria canhave additional functionality prior to incorporation into eukaryoticcells, for example by addition of reporter genes to permit imaging ofthe genetically modified magnetic bacteria and cells labeled with thegenetically modified magnetic bacteria using multimodal imaging. Theeukaryotic cell comprising the multimodal probes can comprise any of thecells described above, including plant cells and animal cells. In someembodiments, the eukaryotic cell comprising the multimodal probes is amammalian cell. In some embodiments, the mammalian cell can comprise acell of a mouse (murine), rat, rabbit, hamster, human, porcine, bovine,or canine. In some embodiments, the mammalian cell is a cancer cell orstem cell, including specific cancer cells, stem cells, and embryoniccells described in the present disclosure.

Accordingly, in some embodiments, a eukaryotic cell can comprisemembrane-enclosed magnetosomes and at least one expressed reporter. Insome embodiments, the eukaryotic cell can comprise a magnetic bacteria,where the bacteria are capable of expressing or expresses one or morereporters. In some embodiments, the eukaryotic cell can comprise amagnetotactic bacteria, where the bacteria are capable of expressing orexpresses one or more reporters. In some embodiments, the reporter isselected from: a fluorescent reporter, a bioluminescent reporter, aPositron Emission Tomography (PET) reporter, Single Photon EmissionComputed Tomography (SPECT) reporter, X-Ray reporter, photoacousticreporter, and an ultrasound reporter, as described herein.

In some embodiments, the eukaryotic cell comprises a magnetic bacteria,where the bacteria express at least two or more reporters. In someembodiments, the two or more reporters are selected from a fluorescentreporter, a bioluminescence reporter, a Positron Emission Tomography(PET) reporter, Single Photon Emission Computed Tomography (SPECT)reporter, X-Ray reporter, photoacoustic reporter, and an ultrasoundreporter.

In some embodiments, the eukaryotic cell comprises a magnetic bacteria,where the bacteria expresses at least three or more reporters. In someembodiments, the three or more reporters are selected from a fluorescentreporter, a bioluminescence reporter, a Positron Emission Tomography(PET) reporter, Single Photon Emission Computed Tomography (SPECT)reporter, X-Ray reporter, photoacoustic reporter, and an ultrasoundreporter.

In some embodiments, the eukaryotic cell comprises a magnetic bacteria,where the bacteria expresses at least one reporter selected from: aPositron Emission Tomography (PET) reporter, Single Photon EmissionComputed Tomography (SPECT) reporter, X-Ray reporter, photoacousticreporter, and an ultrasound reporter. In some embodiments, themagnetotactic bacteria expressing one or more of the foregoingreporters, can also express a fluorescent reporter, a bioluminescentreporter, or both a fluorescent and bioluminescent reporter.

In some embodiments, the eukaryotic cell comprises a single-celledorganism, where the eukaryotic cell is detectable by multiple imagingmodalities. The imaging modalities can be selected from ultrasoundimaging, computed tomography imaging, optical imaging, magneticresonance imaging, optical coherence tomography imaging, radiographyimaging, nuclear medical imaging, positron emission tomography imaging,tomography imaging, photo acoustic tomography imaging, x-ray imaging,thermal imaging, fluoroscopy imaging, bioluminescent imaging, andfluorescent imaging, magnetic particle imaging, and magnetic resonancespectroscopy. In some embodiments, the eukaryotic cell is detectable byat least two or more, three or more, four or more, or five or more ofthe foregoing imaging modalities.

In some embodiments, the eukaryotic cell is detectable by multiplemodalities, where at least one of the modalities is magnetic resonanceimaging. That is, the eukaryotic cell is detectable by a first modality,where the first modality is magnetic resonance imaging. In someembodiments, the eukaryotic cell is detectable by a second modalityselected from ultrasound imaging, computed tomography imaging, opticalimaging, optical coherence tomography imaging, radiography imaging,nuclear medical imaging, positron emission tomography imaging,single-photon emission computerized tomography, tomography imaging,photoacoustic tomography imaging, X-ray imaging, thermal imaging,fluoroscopy imaging, bioluminescent imaging, and fluorescent imaging,magnetic particle imaging, and magnetic resonance spectroscopy.

In some embodiments, the eukaryotic cell is detectable by multiplemodalities, where at least one of the modalities is magnetic particleimaging. In other words, the eukaryotic cell is detectable by a firstmodality, where the first modality is magnetic particle imaging. In someembodiments, the eukaryotic cell is detectable by a second modalityselected from ultrasound imaging, computed tomography imaging, opticalimaging, optical coherence tomography imaging, radiography imaging,nuclear medical imaging, positron emission tomography imaging,single-photon emission computerized tomography, tomography imaging,photoacoustic tomography imaging, X-ray imaging, thermal imaging,fluoroscopy imaging, bioluminescent imaging, fluorescent imaging, andmagnetic resonance spectroscopy.

In some embodiments, where the eukaryotic cell is detectable by magneticresonance imaging or magnetic particle imaging, the second modality isfluorescent imaging. In some embodiments, the second modality isbioluminescent imaging. In some embodiments, the second modality is PETimaging. In some embodiments, the second modality is photoacousticimaging. In some embodiments, the second modality is X-ray imaging. Insome embodiments, the second modality is ultrasound imaging. In someembodiments, the eukaryotic cell is detectable by a third modality, afourth modality, fifth modality, sixth modality or more modalities,where each modality is different.

While use of magnetic bacteria expressing one or more reporter genesprovides flexibility, in some embodiments, the eukaryotic cell itselfcan also express one or more reporters. In particular, the eukaryoticcell comprises a magnetic bacteria, where the eukaryotic cell expressesone or more reporters. Any of the reporters described herein can beexpressed in the eukaryotic cell comprising the magnetic bacteria. Insome embodiments, the eukaryotic cell can express a reporter selectedfrom a fluorescent reporter, a bioluminescent reporter, a PositronEmission Tomography (PET) reporter, Single Photon Emission ComputedTomography (SPECT) reporter, X-Ray reporter, photoacoustic reporter, andan ultrasound reporter.

In some embodiments of the eukaryotic cell comprising a magnetotacticbacteria, the eukaryotic cell can express one or more reporters, and themagnetotactic bacteria can express one or more reporters. In someembodiments, the set of reporters expressed by the eukaryotic cell isdifferent from the set of reporters expressed by the magnetic bacteria.In some embodiments, expression of different reporters by the eukaryoticcell and the magnetotactic bacteria provides a method of distinguishingthe eukaryotic cell from the eukaryotic cell containing themagnetotactic bacteria.

In the embodiments herein, the multimodal probes can be used to imageany cell, tissue, or organism containing the multimodal probe. Inparticular, the multimodal probes can be introduced into eukaryoticcells, e.g., as artificial endosymbionts, and the eukaryotic cell imagedby any of the methods described herein. The eukaryotic cell comprisingthe multimodal probes can be imaged in isolation or as part of a tissueor organism.

Accordingly, in some embodiments, the compositions can be used in amultimodal imaging method comprising: (a) magnetically imaging amagnetotactic bacterium expressing one or more heterologous reporters,and (b) imaging the reporter using a non-magnetic imaging modality.

In some embodiments, the multimodal imaging method can comprise: (a)magnetically imaging a eukaryotic cell comprising a magnetotacticbacterium, and (b) imaging the eukaryotic cell by imaging one or moreexpressed reporters using at least one non-magnetic imaging modality.Any of the reporters described in the present disclosure can be used inthe imaging methods. These include a reporter selected from afluorescent reporter, a bioluminescent reporter, a Positron EmissionTomography (PET) reporter, Single Photon Emission Computed Tomography(SPECT) reporter, X-Ray reporter, photoacoustic reporter, and anultrasound reporter.

In some embodiments, the eukaryotic cell can be multimodally detectedusing magnetic imaging, for example magnetic resonance imaging ormagnetic particle imaging, and an appropriate optical imaging technique.In some embodiments, an optical imaging technique is based on expressionof a fluorescent reporter, for example green fluorescent protein oractive variants thereof.

In some embodiments, the eukaryotic cell can be multimodally detectedusing magnetic imaging, for example magnetic resonance imaging ormagnetic particle imaging, and an appropriate PET imaging technique. Insome embodiments, the PET imaging technique is based on expression of aPET reporter, for example thymidine kinase or cytidine kinase. In thisembodiment, the eukaryotic cell can be multimodally detected using MRIand PET imaging.

In some embodiments, the eukaryotic cell can be multimodally detectedusing magnetic imaging, for example magnetic resonance imaging ormagnetic particle imaging, and an appropriate photoacoustic imagingtechnique. In some embodiments, the photoacoustic imaging technique isbased on expression of a photoacoustic reporter, for example tyrosinaseor β-galactosidase. In this embodiment, the eukaryotic cell can bemultimodally detected using MRI and photoacoustic imaging.

As discussed herein, the multimodal probes and imaging techniques can beused to image eukaryotic cells, including eukaryotic cells that are partof tissues or multicellular organisms. In some embodiments, a multimodalimaging method for imaging a tissue or multicellular organism,comprises: (a) imaging a eukaryotic cell comprising a magnetotacticbacterium by a first imaging modality, wherein the eukaryotic cell is acomponent of a tissue or multicellular organism and the magnetotacticbacterium expresses one or more heterologous reporters, and wherein thefirst imaging modality is a magnetic modality; (b) optionally imagingthe eukaryotic cell by imaging one or more of the expressed reportersusing a second imaging modality, wherein the second modality is anon-magnetic modality; and (c) imaging the tissue or organism by a thirdimaging modality. In some embodiments, the eukaryotic cell is imagedaccording to step (b). As will be apparent to the skilled artisan, insome embodiments, the second imaging modality encompasses one or moreimaging methods, i.e., modalities, in view of the multiple reportersthat can be expressed in the magnetotactic bacteria.

In some embodiments of the method of imaging a tissue or multicellularorganism, the first imaging modality is magnetic particle imaging ormagnetic resonance imaging. In some embodiments, the second imagingmodality comprises a non-magnetic imaging modality selected fromultrasound imaging, computed tomography imaging, optical imaging,optical coherence tomography imaging, radiography imaging, nuclearmedical imaging, positron emission tomography imaging, tomographyimaging, photoacoustic tomography imaging, x-ray imaging, thermalimaging, fluoroscopy imaging, bioluminescent imaging, and fluorescentimaging.

In some embodiments of the method of imaging a tissue or multicellularorganism, the third imaging modality is different from the first imagingmodality. In some embodiments, the third imaging modality is anon-magnetic imaging modality. In some embodiments, the third imagingmodality is selected from ultrasound imaging, computed tomographyimaging, optical imaging, optical coherence tomography imaging,radiography imaging, nuclear medical imaging, positron emissiontomography imaging, tomography imaging, photoacoustic tomographyimaging, X-ray imaging, thermal imaging, fluoroscopy imaging,bioluminescent imaging, and fluorescent imaging.

In some embodiments, multimodality is used for non-invasive in vivoimaging. Generally, imaging modalities can be described as eitheranatomic or functional in terms of the information they provide.Anatomic modalities include X-ray Computed Tomography (CT), MagneticResonance Imaging (MRI) and Ultrasound (US), and these describe variousaspects of the subject anatomy (e.g., variations in material density forCT, proton environments for MRI, and acoustic reflectivity for US).Functional modalities include Positron Emission Tomography (PET), SinglePhoton Emission Computed Tomography (SPECT), Fluorescence Imaging (FLI)and Bioluminescence Imaging (BLI), certain types of MRI (such asDiffusion Weighted (DW) or Dynamic Contrast Enhanced (DCE)), MagneticParticle Imaging (MPI), Photoacoustic Imaging (PAI), contrast-enhancedUltrasound (ceUS), Raman Imaging (RI), contrast-enhanced CT (ceCT) andX-Ray Fluorescence CT (XFCT) (Kuang, Y. et al., “First demonstration ofmultiplexed X-ray fluorescence computed tomography (XFCT) imaging,” IEEETrans Med Imaging 32:262-267 (2012), which is incorporated by referencein its entirety for all purposes), describe specific biologicalprocesses occurring within host cells (including the single-celledorganism) (James, M. L. et al., “A molecular imaging primer: modalities,imaging agents, and applications,” Physiol Rev. 92:897-965 (2012);Molecular Imaging: Principles and Practice, Eds. Weissleder, R., Ross,B. D., Rehemtulla A. and Gambhir, S. S., People's Medical PublishingHouse, USA, (2010); each publication incorporated herein by reference intheir entirety for all purposes). This occurs because the signal beingmeasured is produced by a specific probe molecule which can be achemical (a contrast agent) or protein (a reporter protein). Contrastagents are exogenous and are administered to the subject before thescan, whilst reporter proteins are produced by reporter genes which aregenetically encoded into host cells or the single-celled organism.

In some embodiments, multimodal imaging of the present disclosure isused to track eukaryotic cells in vivo. In order to track cells formultiple generations of eukaryotic cells, the probe needs to be able tomaintain a detectable concentration as the eukaryotic cell divides.Contrast agent methods, which include the nanoparticle approachdescribed above, generally do not satisfy this requirement as theyrapidly dilute to undetectable levels after a few rounds of celldivision. Reporter genes expressing detectable reporters are able tomaintain persistent detectable levels, but require extensive geneticengineering of the eukaryotic cell. In addition, there exist few usefulreporter gene options for long term cell imaging with MRI. Researchershave experimented with MRI reporter genes using the ferritin heavy chaingene (see, e.g., Cohen, B. et al., “Ferritin as an endogenous MRIreporter for noninvasive imaging of gene expression in C6 gliomatumors,” Neoplasia 7:109-117 (2005), incorporated herein by reference inits entirety for all purposes), and with genes such as MagA derived frommagnetotactic bacteria (Goldhawk D. E. et al., “Magnetic resonanceimaging of cells overexpressing MagA, an endogenous contrast agent forlive cell imaging,” Mol Imaging 8:129-139 (2009); Zurkiya, O. et al.,“MagA is sufficient for producing magnetic nanoparticles in mammaliancells, making it an MRI reporter,” Magnet Reson Med. 59:1225-1231(2008), each of which are incorporated herein by reference in theirentirety for all purposes), but the contrast obtainable from thisapproach is typically weak, and provides poor detectability. Anadvantage of multimodal probes described herein, e.g., magnetic bacteriaexpressing one or more reporters, is that the genetically modifiedmagnetic bacteria can be readily introduced into a eukaryotic cell, andthe magnetic bacteria in the eukaryotic cells are heritable to daughtercells such that reporter levels can be maintained.

In some embodiments for multimodal imaging, the multimodal probe or theeukaryotic cell comprising the multimodal probe is contacted with anappropriate reporter probe under suitable conditions to detect presenceof the corresponding expressed reporter, for example a PET, SPECT,photoacoustic, X-ray, or ultrasound reporter. Where the tracking ofcells or imaging is in a tissue or in vivo in an animal, the tissue orthe animal can be administered the appropriate reporter probe to detectand/or image expression of the corresponding reporter. Exemplaryreporter probes for corresponding reporters and the imaging methods havebeen described above. The reporter probes can be administered to thetissue or animal host by various routes. In some embodiments, suitablereporter probes can be administered orally or parenterally. Parenteraladministration in this respect includes administration by intravenous,intramuscular, subcutaneous, intraocular, intrasynovial,transepithelial, including transdermal, ophthalmic, sublingual andbuccal; topically including ophthalmic, dermal, ocular, rectal and nasalinhalation via insufilation, aerosol and rectal systemic. The reporterprobes are administered in a variety of forms, including formulationswith pharmaceutically acceptable excipients or carriers. Thepharmaceutically acceptable excipients and carriers are substantiallynon-toxic and of pharmaceutically acceptable purity. In someembodiments, the forms suitable for injection can include, for example,sterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In some embodiments, the reporter probes can be preparedfor oral administration, for example as orally administered solutions,tablets, capsules or powders.

In some embodiments, as discussed above, multimodal imaging can beperformed in several ways. The first is to image a subject or targetusing different imaging modalities, thus acquiring multiple image typeswhich are then aligned to enable a comparison of the different imagesacross each imaged part of the subject. In some embodiments, anexemplary combination is an anatomic scan (e.g., CT) and a functionalscan (e.g., PET), as this allows the functional data to be placed withinan anatomic context. In some embodiments, multimodal scanners can beused for the acquisition of two or more image types at the same time.Two commonly used examples are combined PET/CT scanners, optical imagerswhich permit the acquisition of FLI, BLI and widefield images, and USscanners which also perform Photo-Acoustic Imaging (PAI). It is to beunderstood that acquisition of many different combinations of imagetypes are possible.

In some embodiments, multimodal imaging can be done by using a probewhich produces an observable signal in two or more imaging modalities.This has the advantage that the imager can use a range of imagingmethods to image the probe and permits greater flexibility in designingimaging studies, as the multimodal probe can be used to introducedesired phenotypes into a subject or target, permitting use of desiredimaging modalities of the subject or target. To this end, in someembodiments, the multimodal probe can express at least two reporters,for example by use of a double fusion reporter construct expressing tworeporter genes, enabling use of at least two imaging modalities. In someembodiments, the multimodal probe can express at least three reporters,for example by use of a triple fusion reporter construct expressingthree reporter genes, which enables use of at least three imagingmodalities, for example, FLI, BLI and PET to image cells which have beenengineered to express it. In some embodiments, the probe can be a singleprobe which produces an observable signal in two or more imagingmodalities. An exemplary probe capable of being imaged with two or moreimaging modalities is reporter tysosinase, which can be imaged usingPET, MRI and photoacoustic imaging techniques. In view of the guidanceherein, numerous variations on this approach can be made and are to beexplicitly described in the present disclosure.

In some embodiments, multimodal images are collected and include atleast two of the following types of images: one or more imagescorresponding to light emitted from the cell, one or more imagescorresponding to light transmitted by the cell, and one or more imagescorresponding to light scattered by the cell. Such multimode imaging canencompass any of the following types of images or combinations: (1) oneor more fluorescent images and at least one bright field image; (2) oneor more fluorescent images and at least one dark field image; (3) one ormore fluorescent images, a bright field image, and a dark field image;and (4) a bright field image. Simultaneous collection of a plurality ofdifferent fluorescent images (separated by spectrum) can also bebeneficial, as well as simultaneous collection of a plurality ofdifferent bright field images (using transmitted light with twodifferent spectral filters). Preferably, the multimode images arecollected simultaneously.

In some embodiments, multimodal imaging can compensate for very lowsignal by combining the low signal with anatomical imaging data that isspatially registered with the low signal from another modality ofdetection. In some embodiments, different imaging methods (ormodalities) allow the visualization of different aspects of theeukaryotic cell and its environment, and combining these multiplemodalities allows one to learn more about the eukaryotic cell and itsenvironment.

In some embodiments, the image acquired by any one modality in any ofthe methods above can be processed in a variety of ways, for examplecomparing and reconstructing the image data. In some embodiments, oneimage obtained using one modality can be compared to image obtainedusing a different modality. In some embodiments, the comparison and/orreconstruction can be carried out by overlaying image acquired from onemodality to the image acquired from a different modality. Comparisonsand/or reconstructions can be carried out for 2 or more images, 3 ormore images, 4 or more images, 5 or more images, or 6 or more images,where each image is acquired using different modalities. In general, theimages are in the form of pixel images. For example, in the multimodalimaging method, comprising: (a) magnetically imaging a magnetotacticbacterium expressing one or more heterologous reporters, and (b) imagingthe reporter using a non-magnetic modality, the method can furthercomprise overlaying to each other the image acquired/captured from (a)and the image acquired/captured from (b). In some embodiments, the imagein (a) and the image in (b) comprise pixel images. Various methods forimage processing, including comparing or reconstructing images obtainedby different modalities are described in, for example, U.S. patentpublication Nos. 20110262016 and 20130177224.

In addition to the advantage of the heritability of magnetic bacteria ineukaryotic cells, which overcomes the need to construct eukaryotic cellsexpressing a reporter while maintaining levels of the reporter fordetection, use of multimodal imaging based on magnetic bacteria as amultimodal probe allows enhanced imaging of localization and tracking ofeukaryotic cells, including imaging of changes in cell dynamics, such aswithin tissues containing the eukaryotic cells. This added dimensionenhances the ability to track cells and tissues containing such cells.

For example, in some embodiments, the multimodal probes can beintroduced into cancer cells, and the modified cancer cells introducedinto experimental animals, for example to track metastasis and movementof cancer cells in the animal. The cancer cells can also be tracked andimaged following various therapeutic treatments to assess thetreatment's effectiveness and examine the dynamics of cancer cellviability and migration before and after treatment.

In some embodiments, the multimodal probes can be introduced into stemcells to track the stem cell's in vivo fate, including localization,growth rate and viability. Where the stem cells are hematopoietic stemor progenitor cells, the stem cells containing the multimodal probes areintroduced into a mammal and the behavior of the magnetic hematopoieticstem or progenitor cells, including their localization, proliferationand mobilization into blood stream upon receiving different stimuli,monitored through multimodal imaging. This can be coupled to differenttypes of treatments meant to facilitate stem cell transplantation andhoming. Other uses of the multimodal imaging methods will be apparent inview of the guidance provided in the present disclosure.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter.

EXAMPLES Example 1 Microinjection of gfp⁺AMB-1 into Murine Cells

A. Construction of gfp⁺AMB-1.

Expression vectors for eGFP, one including a Shine-Dalgarno sequenceupstream of the gfp gene and one without a Shine Dalgarno, sequence werecloned into cryptic broad host range vector pBBR1MCS-2 (Kovach, M. E. etal., “Four new derivatives of the broad-host-range cloning vectorpBBR1MCS, carrying different antibiotic-resistance cassettes,” Gene 166,175-176, (1995), incorporated herein by reference in its entirety forall purposes). AMB-1 (ATCC 700264) was transformed with this construct(see, e.g., Matsunaga, T. et al., “Complete genome sequence of thefacultative anaerobic magnetotactic bacterium Magnetospirillum sp.strain AMB-1,” DNA Res. 12, 157-166 (2005); Burgess J. G., et al.,“Evolutionary relationships among Magnetospirillum strains inferred fromphylogenetic analysis of 16S rDNA sequences,” J Bacteriol. 175:6689-6694(1993); Matsunaga T, et al., “Gene transfer in magnetic bacteria:transposon mutagenesis and cloning of genomic DNA fragments required formagnetosome synthesis,” J Bacteriol. 174: 2748-2753 (1992); Kawaguchi R,et al., “Phylogeny and 16s rRNA sequence of Magnetospirillum sp. AMB-1,an aerobic magnetic bacterium,” Nucleic Acids Res. 20:1140 (1992); eachpublication incorporated herein by reference in its entirety for allpurposes).

Transformation was achieved by conjugation using a donor Escherichiacoli strain (see Goulian, M. et al., “A simple system for convertinglacZ to gfp reporter fusions in diverse bacteria,” Gene 372:219-226(2006); Scheffel, A. Schiller, D., “The Acidic Repetitive Domain of theMagnetospirillum gryphiswaldense MamJ Protein Displays Hypervariabilitybut Is Not Required for Magnetosome Chain Assembly,” J Bacteriol.189(17):6437-6446 (2007); each publication incorporated herein byreference in its entirety for all purposes). The mating reactions werecultured for 10 days under defined microaerophilic conditions in theabsence of DAP to select for positive transformants.

Following conjugation, gfp⁺AMB-1 transformants with and without theShine-Dalgarno sequence successfully displayed GFP fluorescence. Thetransformants containing the Shine-Dalgarno sequence displayed higherlevels of GFP fluorescence than the transformants without this sequence.The resulting fluorescence did not leave the gfp⁺AMB-1 cells when viewedat 100× magnification at 488 nm excitation.

The magnetic properties of the gfp⁺AMB-1 were analyzed by MRI. Thegfp⁻AMB-1 were suspended in agar plugs using a 1.5T instrument tooptimize and characterize the imaging properties. FIG. 1 shows thepositive contrast generated with a T₁ pulse sequence over a log scaleconcentration up to ˜10⁸ MTB/mL. Signal intensity was related toconcentration.

B. Microinjection into Murine Embryonic Cells.

The gfp⁺AMB-1 were microinjected into one cell of each of 170 mouseembryos at the 2-cell stage. Six concentrations over a log scale up to˜10⁵ gfp⁺AMB-1 were injected per cell, estimated by the optical densityat 565 nm. Death rate of cells following microinjection was constantacross the different injected concentrations. Images overlayingfluorescent and differential interference contrast (DIC) images of cellsinjected with the highest concentration (10⁵ MTB/cell) were compared. Anuninjected control exhibited low levels of autofluorescence. Slices atdifferent horizontal planes in 8-cell embryos at a given time point werecompared. In each embryo, all four cells derived from the injected cellshowed significant fluorescence while none of the four cells derivedfrom the uninjected internal controls displayed significantfluorescence.

The embryos were allowed to develop for three days after the injection.In each concentration level, embryos survived for up to the full threedays developing to the 256 cell blastula stage and appeared healthyenough for implantation. Numerous cells within each blastula displayedsignificant fluorescence, demonstrating that the artificialendosymbionts were transferred to daughter cells across multiple celldivisions as the embryos comprising the eukaryotic host cells developedto the blastula stage. One such blastula is shown in FIG. 2, where panelA shows a differential interference contrast (DIC) image of the blastulaand panel B) shows a gray scale fluorescence capture of the same image,showing fluorescence in numerous cells throughout the blastula.

Confocal microscopy was used to quantify total expression of GFPthroughout four individual embryos by measuring total GFP fluorescencein the entire embryo over time at various points beginning at the eightcell stage of the embryo. FIG. 3 shows the change of embryo fluorescenceover time. This indicates that the copy number of artificialendosymbionts was maintained in daughter cells for at least sevengenerations, such that the fluorescent phenotype of the host cells wasmaintained as the embryo progressed from the 2-cell stage to the256-cell blastula stage.

These results demonstrate that, when delivered by microinjection,gfp⁺AMB-1 were not immediately cleared or degraded and were not toxic tothe developing embryo over the course of the three day experiment.Microinjected embryos divided normally, suggesting that gfp⁺AMB-1 do notdisplay pathogenic markers or secret toxic compounds. They weretransferred to daughter cells across many cell divisions, were containedin the cytoplasm, were punctate and well distributed, and maintainedcopy number within the daughter host cells, such that the fluorescentphenotype of the eukaryote host cells was maintained in daughter cellsthrough at least seven generations. These results demonstrate that AMB-1can be stably maintained intracellularly and are transferred to daughtercells over at least seven cell divisions.

Example 2 Phagocytic Entry of AMB-1

Receptor mediated: The inlAB gene is amplified from L. monocytogenesgenomic DNA (ATCC 19114) and is inserted into pBBR1MCS-5, the gentamicincognate of pBBR1MCS-2 (see Kovach, M. E., et al., “Four new derivativesof the broad-host-range cloning vector pBBR1MCS, carrying differentantibiotic-resistance cassettes,” Gene 166, 175-176, (1995)), andgfp+inlAB+ AMB-1 is generated. The gfp+inlAB+ AMB-1 is co-cultured witheukaryotic host cells, including common epithelial tumor cell linesCoco-2, MDA-MB-231 and MCF7, non-epithelial tumor cell lines, such asHT-1080 and HL60, and murine stem cells. Fluorescent microscopy and FACSare used to monitor and quantify internalization and intracellularlocation.

Expression of pore-forming haemolysin (hlyA) in AMB-1 is achievedthrough amplification of hlyA from L. monocytogenes genomic DNA (ATCC19114). The amplified hlyA is inserted into pBBR1MCS-3 (the tetracyclinecognate of pBBR1MCS-2), which is then used to transform gfp⁺AMB-1. Theresulting AMB-1 strain is exposed to murine macrophage cell line J774,capable of spontaneous phagocytosis. Gentomycin treatment is used toeliminate bacteria not internalized and hlyA⁻AMB-1 is used as negativecontrol. Fluorescent microscopy is used to monitor the intracellularfate and localization of AMB-1.

If bacteria remain confined to the phagosomes, two genes, plcA and plcB,implicated in escape of L. monocytogenes into the cytosol, areintroduced. (Smith, G. A. et al., Infection and immunity 63:4231 (1995);Camilli, A. et al., J Exp Med. 173:751 (1991); each publicationincorporated herein by reference in its entirety for all purposes). Ifbacteria escape successfully, but fail to propagate, hpt is introduced(see Goetz, M. et al., Proc Natl Acad Sci USA 98:12221 (2001);Chico-Calero, I. et al., Proc Natl Acad Sci USA 99:431 (2002); eachpublication incorporated herein by reference in its entirety for allpurposes). In L. monocytogenes, hpt encodes the transporter responsiblefor uptake of glucose-6-phosphate from the cytosol. Other genes from L.monocytogenes have been implicated in sustaining growth within host(glnA and gltAB and argD) and these are systematically introduced asneeded. (Joseph, B. et al., J Bacteriol. 188:556 (2006), incorporatedherein by reference in its entirety for all purposes).

Example 3 Regulation of AMB-1 Growth

Regulation of AMB-1 growth in embryonic stem cells can be regulated asfollows. Coleoptericin-A (ColA) is amplified from total Sitophilusoryzae cDNA. Expression of ColA in beetles of genus Sitophilus regulatestiters of γ-Protobacterium, which has naturally developed closesymbiotic relationship the beetles, and resides in specific cells calledbacteriocytes. (Login, F. H. et al., “Antimicrobial peptides keep insectendosymbionts under control,” Science 334(6054):362-365 (2011),incorporated herein by reference in its entirety for all purposes).

Murine embryonic stem cells comprising gfp+AMB-1 are treated using aneural differentiation protocol. MTB expression levels are quantifiedusing qPCR and fluorescent microscopy. Amplified colA is then expressedin the gfp+AMB-1 embryonic stem cells. A promoter is selected to provideoptimal ColA expression levels.

Example 4 Magnetic Phenotype of Murine Cells Containing gfp⁺AMB-1

Cells from macrophage cell line J774.2 derived from murine ascites andsolid tumor with introduced gfp⁺AMB-1 were applied to a magnetic columnand were retained by the column. These results demonstrate that,following introduction of gfp⁺AMB-1, J774.2 murine cells weremagnetically detected and magnetically manipulated, as they weremagnetically concentrated and magnetically collected.

Example 5 Gfp⁺AMB-1 in Human Breast Cancer Cell Line MDA-MB-231

Gfp⁺AMB-1, Gfp+InlA/B+AMB-1, and Gfp+Pla1+AMB-1 were each introduced tohuman breast cancer cells from cell line MDA-MB-231. GFP fluorescencewas detected in more than 90% of the MDA-MB-231 cells 48 hours after theintroduction of each of Gfp⁺AMB-1, Gfp+InlA/B+AMB-1, and Gfp+Pla1+AMB-1.GFP fluorescence in Gfp⁺AMB-1 was observed in these MDA-MB-231 cells atleast 13 days after introduction of geAMB-1, in the fourth passage ofthe MDA-MB-231 cells following the introduction, where MDA-MB-231 cellpopulation doubled three to four times between each passage. GFPfluorescence was observed in both forming daughter cells of anMDA-MB-231 cell with introduced geAMB-1 in the process of cell division.

Following the introduction of gfp⁺AMB-1, MDA-MB-231 cells were stainedwith Lysotracker® Red DND-99 dye (specific to lysosomes) and Hoechst33342 nuclear stain purchased from Life Technologies. Green GFPfluorescence was observed as localized within individual MDA-MB-231cells and distinct from red lysosome staining and blue nuclear stainingsuggesting that AMB-1 were localized in cytoplasm and not digestedthrough lysosome pathway.

At 24 hours and 72 hours after introduction, plated MDA-MB-231 cells andplated control MDA-MB-231 cells were fixed in formalin andglutaraldehyde following a wash with PBS. Cells were then stained withPrussian Blue, and observed by microscopy at 40× magnification. Ironstaining was observed in some of the MDA-MB-231 cells with introducedgfp⁺AMB-1 but not in the control MDA-MB-231 cells without introducedgfp⁺AMB-1. The proportion of cells displaying iron staining was similarbetween the MDA-MB-231 cells 72 hours after gfp⁺AMB-1 introduction andthe MDA-MB-231 cells 24 hours after gfp⁺AMB-1 introduction.

48 hours after the introduction of gfp⁺AMB-1, MDA-MB-231 cells weretrypsinized and resuspended in PBS. One sample of these cells was placedinto a glass slide chamber. A magnet was aligned to the side of chamberand cell movement was observed under microscope at 20× magnification.MDA-MB-231 cells with introduced geAMB-1, but not control MDA-MB-231cells which had not had geAMB-1 introduced, exhibited movement towardthe magnet. Another sample of these cells was placed into small tubes,which were taped to a magnet for one hour. Control MDA-MB-231 cellswhich had not had gfp⁺AMB-1 introduced settled down at the bottom of thetube. However, MDA-MB-231 cells with introduced geAMB-1 were aligned tothe magnet side of the tubes.

These results indicate that gfp⁺AMB-1 were not immediately cleared fromhuman breast cancer MDA-MB-23 1 cells. They were transferred to daughtercells across at least 12 cell divisions and were located within theMDA-MB-231 cells outside of both the lysosomes and nuclei. These resultsalso demonstrate that at least 48 hours following introduction ofgfp⁺AMB-1, the MDA-MB-231 cells containing gfp⁺AMB-1 displayed weremagnetically detected and magnetically manipulated, as they weremagnetically moved, magnetically targeted to a location, magneticallyconcentrated, and magnetically collected. Additionally, at least 72hours following introduction of gfp⁺AMB-1, the MDA-MB-231 cellscontained observable quantities of iron.

Example 6 Gfp⁺AMB-1 in Human Induced Pluripotent Stem Cells

GFP fluorescence was observed in Human Induced Pluripotent Stem (“IPS”)cells at least eight days following introduction of gfp⁺AMB-1 to the IPScells, in the second passage of the IPS cells. These results indicatethat gfp⁺AMB-1 were not immediately cleared from human IPS cells, andwere transferred to daughter cells.

Example 7 Cell Imaging within Mouse Tumor

Cell visualization was tested in a mouse bearing two subcutaneoustumors, one on its left flank and one on its right flank. 1.5×10⁶MDA-MB-231 cells containing introduced gfp⁺AMB-1 were injected directlyinto the tumor on the left flank of the mouse. An equivalent number ofcontrol MDA-MB-231 cells without introduced cells were injected on theright flank of the mouse. The mouse was imaged using a bench top 1T MRIwith T2w pulse sequences. The resulting image showed a dark area at thetumor on the right side of the mouse, the site of the injection ofMDA-MB-231 cells containing introduced geAMB-1, but no signal at thetumor on the right side of the mouse, where control MDA-MB-231 cellsinjected into a left side tumor.

Example 8 Monitoring of Mouse Tumor

Gfp⁺AMB-1 cells are introduced into MDA-MB-231 human cancer cells. Theresulting magnetic cells and their daughter cells are injected intomammary fat pads of a group of immunocompromised mice. Tumor growth ismonitored at regular intervals by MRI imaging. Mice with establishedtumors assigned either to an experimental group or to a control group.Mice in the experimental group are treated with a potential anti-tumortherapeutic compound while mice in the control group are treated with aninactive vehicle. MRI is used to monitor the size and growth of thetumor non-invasively following the treatments to assess the efficacy ofthe tested compound in combating the tumor.

Example 9 Magnetic Enhancement of Cell Retention

Gfp⁺AMB-1 cells are introduced into Rat Cardiac-Derived Stem Cells(CDC). The resulting magnetic CDC cells are used in theIschemia/Reperfusion model. Rats are treated as described in Cheng K. etal., “Magnetic enhancement of cell retention, engraftment, andfunctional benefit after intracoronary delivery of cardiac-derived stemcells in a rat model of ischemia/reperfusion,” Cell Transplant.21(6):1121-35 (2012). The magnetic CDC cells are then introduced intothe left ventricle cavity of the treated rates. A 1.3 T magnet is placedabove the heart during and after the injection. The animal's chest isclosed and it is allowed to recover. The short and long-term behavior ofthe labeled CDC in the rat is monitored by MRI imaging at regularintervals.

Example 10 AMF Tumor Treatment

Gfp⁺AMB-1 cells are introduced into MDA-MB-231 cells. The resultingcells are injected into subcutaneous tumors, formed by 4T1 cells in nudemice. Untreated control MDA-MB-231 cells are injected into a tumor atthe opposite flank of each animal. Each animal is placed intoalternating magnetic field (30.6 kA/m, 118 kHz) for 30 minutes, andallowed to recover following the procedure. Animals are sacrificed atregular intervals and histological analyzes are performed on tumors fromboth mice with injected magnetic MDA-MB-231 and control mice withinjected control MDA-MB-231. In the experimental mice, the labeled cellsand the surrounding tumor are damaged leading to damage to the tumorovertime.

Example 11 Bioluminescent AMB-1

Gene sequences of Photorhabdus luminescens luxCDABE operon were PCRamplified from pXen-13 plasmid (Perkin Elmer Hopkinton, Ma.) and clonedinto pBBR1-MCS2 by using In-Fusion® HD Cloning Kit (Clontech, MountainView, Calif., USA). Positive clones were confirmed by showing right sizeinsert by restriction digest analysis and luminescence detection by aluminescence reader. Purified plasmids were introduced to mating strainE. coli WM3064. AMB-1 and WM3064 strains were mated for plasmid transferand kanamycin resistant AMB-1 strains were selected. Luminescencepositive strains were cultured in liquid MG medium. Luminescence wasconfirmed for lux+ AMB-1 alone using a benchtop luminometer. Magneticproperties were confirmed using CMag measurements. Strains wereintroduced into eukaryotic cells through either co-centrifugation orincubation with a strong magnet next to the cell container. Magneticproperties were confirmed by MRI phantoms and also by observing magneticsensitivity in a microscope or by being trapped on a magnetic column.

Example 12 Creation of Fluorescent AMB-1

In an embodiment, genetically modified magnetotactic bacteria detectableby FLI are made using enhanced Green Fluorescent Protein (eGFP) insertedinto the suicide plasmid pJQ200 and introduced into a diaminopimelicacid auxotrophic E. coli strain, which was used for mating with AMB-1.Gentamicin resistant strains were selected and fluorescence wasconfirmed by fluorescent microscopy. After 3 passages in gentamicinmedia, the cells were grown for a further 3 passages without antibioticselection. They were then grown in the presence of 1% sucrose for 6 daysand plated for single colonies on MGB agar plates. Single colonies werechecked for GFP. Detection was performed using a Nikon Ti—S invertedepifluorescence microscope, using a FITC filter and a 40× objective.Fluorescent spiral-shaped bacteria can be observed at 40× magnification.

GFP-positive AMB-1 were inserted into MDA-MB-231, 4T1, hAMSC, J774.2,MCF-7 (and other) cells. gfp+ AMB-1 labeled and unlabeled cells wereobserved using a fluorescence microscope, and a clear enhancement incellular fluorescence was observed with the gfp+ AMB-1 labeled cells,often with a punctate pattern that suggests localization inintracellular vesicles.

FIGS. 6A-L shows confocal microscopy and fluorescence images of the iPScells (pluripotent stem cells) containing gfp+ AMB-1, MDA-MB-231 cells(human breast carcinoma) containing gfp+ AMB-1, J774.2 cells (murinemacrophages) containing gfp+ AMB-1, BJ cells (human fibroblasts)containing gfp+ AMB-1, HEP1 cells (human liver adenocarcinoma)containing gfp+ AMB-1, and MCF-7 cells (human epithelial breastadenocarcinoma) containing gfp+ AMB-1. gfp+ AMB-1 were inserted intothese cell lines and microscopy images were acquired using a Nikon Ti—Sepi-fluorescence microscope with phase contrast optics or a FITCfluorescence filter, with a 40× magnification lens. FIGS. 6A-B showphase contrast and fluorescence images of iPS cells (pluripotent stemcells) containing gfp+ AMB-1. FIGS. 6C-D show phase contrast andfluorescence images of MDA-MB-231 cells (human breast carcinoma)containing gfp+ AMB-1. FIGS. 6E-F show phase contrast and fluorescenceimages of J774.2 cells (murine macrophages) containing gfp+ AMB-1. FIGS.6G-H show phase contrast and fluorescence images of BJ cells (humanfibroblasts) containing gfp+ AMB-1. FIGS. 6I-J show phase contrast andfluorescence images of HEP1 cells (human liver adenocarcinoma)containing gfp+ AMB-1. FIGS. 6K-L show phase contrast and fluorescenceimages of MCF-7 cells (human epithelial breast adenocarcinoma)containing gfp+ AMB-1. All these cell types showed enhanced fluorescencewhen the cells contained gfp+ AMB-1.

Example 13 Creation of PET Detectable AMB-1

To create PET-enabled genetically modified magnetotactic bacteria, thegene sequence for human simplex virus thymidine kinase (HSV1-Tk) can beintroduced via an appropriate plasmid containing a selection gene (e.g.for kanamycin or gentamicin antibiotic resistance) into WM3064, whichcan subsequently be mated with AMB-1 for plasmid transfer.Antibiotic-resistant AMB-1 strains can then be selected. Positiveexpression of the reporter can be evaluated by adding the radiolabelledHSV1-Tk substrate 3H-PCV (penciclovir), and quantifying AMB-1 uptakeusing a scintillation counter.

PET reporter-positive AMB-1 can be inserted into MDA-MB-231, 4T1, hAMSC,J774.2, MCF-7 (and other) cells. AMB-1 labeled and unlabeled cells canbe injected into a living subject, and then the subject would beinjected with a radiolabelled substrate for the HSV1-TK reporter gene(e.g. ¹⁸F-FHBG). After 1 hour, a PET scan of the subject would show apronounced uptake in the region of the AMB-1 labeled cells.

Example 14 Detection of Eukaryotic Cells Containing AMB-1 withFluorescence Imaging and MRI

MDA-MB-231 cells containing gfp+ AMB-1 were grown to confluence.MDA-MB-231 cells containing gfp+ AMB-1 and MDA-MB-231 cells without gfp+AMB-1 were imaged with confocal microscopy and fluorescence imaging.Phase contrast and fluorescence images were acquired using a Nikon Ti—Sepi-fluorescence microscope with phase contrast optics or a FITCfluorescence filter, with a 40× magnification lens. Fluorescence imageswere exposed for 0.5 seconds. FIGS. 4A and 4D show phase contrast imagesof MDA-MB-231 with and without gfp+ AMB-1. FIGS. 4B and 4E showfluorescence imaging of MDA-MB-231 with and without gfp+ AMB-1. FIG. 4Bdemonstrates the fluorescent signal from the gfp+ AMB-1 in theMDA-MB-231 cells.

MDA-MB-231 cells with and without gfp+ AMB-1 were prepared for imagingwith MRI as follows. MDA-MB-231 cells with and without gfp+ AMB-1 weretrypsinized and 2 million cells were resuspended in 100 μl PBS. Cellsuspensions were pipetted into PCR tubes containing pre-solidified 100μl 1% agarose. PCR tubes were sealed and left for 10 minutes to allowcells to settle on the agarose/PBS boundary. A Varian 7T 300 MHzHorizontal Bore preclinical MR system was used. The scanner featured a40 G/cm 120 mm High Duty Cycle Gradient Coil. Images were acquired withvnmrj 4.0 image acquisition software. Phantom tubes were imaged using a2D T2w MEMS sequence (TR 3000 ms, TE Min 7 ms, NE 48, NEX 1, 128×128,40×40 mm FOV, 1 mm slice thickness, coronal view, tubes were imagedusing a Rapid F19 volume coil. MRI images are shown in FIGS. 4C(MDA-MB-231 with gfp+ AMB-1) and 4F (MDA-MB-231 without gfp+ AMB-1). Adark band is seen in FIG. 4C identifying the location of the MDA-MB-231with gfp+ AMB-1. No such dark band is seen in FIG. 4F.

FIGS. 5A-C shows a lower magnification example of MRI, fluorescence, andvisual inspection imaging of MDA-MB-231 cells with gfp+ AMB-1.MDA-MB-231 cells were labeled with gfp+ AMB-1, washed after 24 hours,trypsinized and harvested at 48 hours, spun down in a centrifuge, andthe resultant pellet was suspended in 1% agarose gel in a cylindrical100 cc tube. Fluorescence images were obtained on an IVIS 49fluorescence imager, using a Cy5.5 filter and a 1 second acquisitiontime. An MRI image was obtained on a Bruker ICON 1T preclinical MRIscanner, with a T2w RARE pulse sequence (TR 1500 ms, TE 96 ms, FA 180degrees, acquisition time 5 mins, FOV 3.5 cm, 128×128, slice thickness1.25 mm, interslice gap 1.00 mm), with a 2.5 cm diameter rat coil. FIGS.5A-C show different modality detection of the same MDA-MB-231 cells withgfp+ AMB-1 suspended in agarose. FIG. 5A shows the MDA-MB-231 cellsviewed by visual inspection of the plastic tube. FIG. 5B shows an MRIimage of the MDA-MB-231 cells with gfp+ AMB-1 in the same tube. FIG. 5Cshows a fluorescence image of the MDA-MB-231 cells in the same tube. TheMDA-MB-231 cells with gfp+ AMB-1 are detectable using MRI andfluorescence imaging.

Example 15 Detection of AMB-1 with Luminescence and MRI

AMB-1 was transfected with a pBBR1-MCS2 lux plasmid to make luminescentAMB-1. These luminescent AMB-1 were introduced into MDA-MB-231 cells.MDA-MB-231 cells contain the lux+AMB-1 were grown to confluence and 1million cells subject to luminosity measurements on a TD-20/20luminometer using the manufactures protocol. Measurements were obtainedfor 1 minute and results were standardized for OD400=1. FIG. 7A shows achart comparing the luminescence signal from the MDA-MB-231 cells withluminescent AMB-1 to MDA-MB-231 cells without AMB-1. MDA-MB-231 cellswith luminescent AMB-1 yielded a significant luminescence signalcompared to MDA-MB-231 cells without AMB-1.

An MRI image of MDA-MB-231 cells with luminescent AMB-1 was obtained asfollows. MDA-MB-231 cells with luminescent AMB-1 were trypsinized and 2million cells were resuspended in 100 μl PBS. Cell suspensions werepipetted into PCR tubes containing pre-solidified 100 μl 1% agarose. PCRtubes were sealed and left for 10 minutes to allow cells to settle onthe agarose/PBS boundary. Images were acquired on a Discovery MR901preclinical 7T MRI, actively shielded; Bore diameter—310 mm, Gradients:(120 mm ID, 600 mT/m, 6000 T/m/s), 8 Receive channels; 1 MHz at 16 bitsper channel. A T2*w GRE sequence was used for imaging sample (TR 300 ms,TE 30 ms, NEX 2, 256×256, 30 mm FOV). FIG. 7B shows an MRI image ofMDA-MB-231 cells with luminescent AMB-1. The MDA-MB-231 cells withluminescent AMB-1 are seen as a dark band in the middle of the tube.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. A method for detecting the location and viability of aeukaryotic cell, comprising the steps of: obtaining the eukaryotic cell,wherein the eukaryotic cell contains a particle of Fe₃O₄; andmagnetically imaging the particle of Fe₃O₄ using a magnetic particleimaging whereby the location and viability of the eukaryotic cell isdetected.
 22. The method of claim 21, wherein the eukaryotic cell iscontained in a mammal.
 23. The method of claim 22, wherein the mammal isa human.
 24. The method of claim 22 wherein the eukaryotic cell is astem cell.
 25. The method of claim 22, wherein the eukaryotic cell is aT-cell.
 26. The method of claim 22, wherein the eukaryotic cell is acancer cell.
 27. The method of claim 22, wherein the magnetic particleimaging generates a MRI image.
 28. The method of claim 27 wherein theMRI image is a T2 MRI image or a T2*MRI image.
 29. The method of claim28, wherein the MRI image is a T2*MRI image.
 30. The method of claim 22,wherein the imaging is done with a CT scanner.
 31. A method fordetecting the location and viability of a eukaryotic cell, comprisingthe steps of: obtaining the eukaryotic cell, wherein the eukaryotic cellcontains a particle of superparamagnetic iron oxide; and magneticallyimaging the particle of superparamagnetic iron oxide using a magneticparticle imaging whereby the location and viability of the eukaryoticcell is detected.
 32. The method of claim 31, wherein the eukaryoticcell is contained in a mammal.
 33. The method of claim 32, wherein themammal is a human.
 34. The method of claim 32 wherein the eukaryoticcell is a stem cell.
 35. The method of claim 32, wherein the eukaryoticcell is a T-cell.
 36. The method of claim 32, wherein the eukaryoticcell is a cancer cell.
 37. The method of claim 32, wherein the magneticparticle imaging generates a MRI image.
 38. The method of claim 37wherein the MRI image is a T2 MRI image or a T2*MRI image.
 39. Themethod of claim 38, wherein the MRI image is a T2*MRI image.
 40. Amethod for detecting the location and viability of a eukaryotic cell,comprising the steps of: obtaining the eukaryotic cell, wherein theeukaryotic cell contains a particle of superparamagnetic iron oxide; andmagnetically imaging the particle of superparamagnetic iron oxide usinga CT scanner whereby the location and viability of the eukaryotic cellis detected.